Accelerated directed evolution of microbial consortia for the development of desirable plant phenotypic traits

ABSTRACT

The disclosure relates to methods for the screening, identification, and/or application of one or more microorganisms of use in imparting one or more beneficial properties to one or more plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 16/686,681 filed Nov. 18, 2019, which is a Continuation Applicationof U.S. application Ser. No. 15/676,594, filed on Aug. 14, 2017 thatissued as U.S. Pat. No. 10,526,599 on Jan. 7, 2020, which is aContinuation Application of U.S. application Ser. No. 15/178,143, filedon Jun. 9, 2016, and that issued as U.S. Pat. No. 9,732,336 on Aug. 15,2017, which is a Continuation Application of U.S. application Ser. No.14/991,543, filed on Jan. 8, 2016, and that issued as U.S. Pat. No.9,365,847 on Jun. 14, 2016, which itself is a Continuation Applicationof U.S. application Ser. No. 14/835,867, filed on Aug. 26, 2015 and thatissued as U.S. Pat. No. 9,260,713 on Feb. 16, 2016, which itself is aContinuation Application of U.S. application Ser. No. 14/218,920, filedon Mar. 18, 2014 and that issued as U.S. Pat. No. 9,150,851 on Oct. 6,2015, which is a U.S. Utility Application under 35 U.S.C. § 1.111(a)that claims priority pursuant to 35 U.S.C. § 120, as aContinuation-in-Part Application, to International Application No.PCT/NZ2013/000171, filed on Sep. 19, 2013, which itself claims priorityto New Zealand Application No. 602532, filed on Sep. 19, 2012.

The entire contents of each of the aforementioned applications andpatents are hereby incorporated by reference in their entirety for allpurposes.

FIELD

The present disclosure relates to methods for the screening,identification, and application of microorganisms of use in impartingbeneficial properties to plants.

In particular aspects, the present disclosure provides for methods ofdeveloping microbial consortia through directed evolution andaccelerated microbial selection. The microbial consortia developed bythe methods of the present disclosure are capable of producing desirableplant phenotypic responses. Other aspects of the disclosure identifyindividual microbes, such as one or more microorganisms.

BACKGROUND

Known processes of imparting beneficial properties to plants, such asselective breeding, can be extremely costly, slow, limited in scope, andfraught with regulatory difficulties. Few commercial successes haveeventuated from over two decades of large-scale investment into thistechnology.

Despite many decades of successful scientific research into theconventional breeding of highly-productive crops and into development oftransgenic crops, relatively little research effort has been directed atdevelopment of plant traits via other means.

Thus, there is a great need in the art for the development of methods toimprove plant traits that do not suffer from the drawbacks associatedwith the present technology.

BRIEF SUMMARY OF THE DISCLOSURE

The disclosure provides for an efficient, fast, and broadly applicableplatform that can be utilized to develop microbes and microbialconsortia that promote one or more desirable plant properties. In someembodiments, a single microbe is identified.

In certain aspects, the disclosure provides for the development ofhighly functional microbial consortia that help promote the developmentof a desired phenotypic or genotypic plant trait.

It is one object of the present disclosure to provide a method for theselection of one or more microorganism or composition of microorganismsthat are of use in imparting one or more beneficial properties to aplant.

It is a further object of the disclosure to provide a system forassisting in the improvement of one or more plants.

Selective-Pressure Independent Accelerated Microbial SelectionMethodology

In a first broad aspect of the present disclosure there is provided amethod for the selection of one or more microorganisms capable ofimparting one or more beneficial properties to a plant, the methodcomprising at least the steps of:

-   -   a) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to a growth        medium in the presence of a first set of one or more        microorganisms;    -   b) selecting one or more plant following step a);    -   c) acquiring a second set of one or more microorganisms        associated with said one or more plant selected in step b) or        plant growth media;    -   d) repeating steps a) to c) one or more times, wherein the        second set of one or more microorganisms acquired in step c) is        used as the first set of microorganisms in step a) of any        successive repeat.

In one embodiment, the second set of one or more microorganisms areisolated from said one or more plant in step c).

In one embodiment, the first set of one or more microorganisms and/orthe second set of one or more microorganisms are selected from themicroorganisms detailed herein after.

In one embodiment, the growth medium is selected from the growth mediadetailed herein after.

In one embodiment, the step of subjecting one or more plant to a growthmedium involves growing or multiplying the plant.

In one embodiment, two or more plants are subjected to a growth mediumin the presence of one or more microorganisms. In other embodiments 1 to10, or 1 to 20, or 1 to 30, or 1 to 40, or 1 to 50, or 1 to 60, or 1 to70, or 1 to 80, or 1 to 90, or 1 to 100, or 1 to 200, or 1 to 500, or 1to 1000, or 1 to 10,000 plants are subjected to a growth medium in thepresence of the first set of microorganisms. In other embodiments, 10 ormore, 20 or more, 100 or more, 300 or more, 500 or more, or 1000 or moreplants are subjected to a growth medium in the presence of the first setof microorganisms.

In one embodiment, the one or more plant is selected (step b) on thebasis of one or more selection criterion.

In one embodiment, the one or more plant is selected on the basis of oneor more phenotypic trait. In one embodiment, the one or more plant isselected based on the presence of a desirable phenotypic trait. In oneembodiment, the phenotypic trait is one of those detailed herein after.

In one embodiment, the one or more plant is selected on the basis of oneor more genotypic trait. In one embodiment, the one or more plant isselected based on the presence of a desirable genotypic trait.

In one embodiment, the one or more plant is selected based on acombination of one or more genotypic and one or more phenotypic traits.In one embodiment, different selection criteria may be used in differentiterations of a method of the disclosure.

In one embodiment, the second set of one or more microorganisms (step c)are isolated from the root, stem and/or foliar (including reproductive)tissue of the one or more plants selected. Alternatively, the second setof one or more microorganisms are isolated from whole plant tissue ofthe one or more plants selected. In another embodiment, the planttissues may be surface sterilised and then one or more microorganismsisolated from any tissue of the one or more plants. This embodimentallows for the targeted selection of endophytic microorganisms. Inanother embodiment, the second set of one or more microorganisms may beisolated from the growth medium surrounding selected plants. In anotherembodiment, the second set of one or more microorganisms are acquired incrude form.

In one embodiment, the one or more microorganisms are acquired in stepc) any time after germination.

In one embodiment, where two or more microorganisms are acquired in stepc), the method further comprises the steps of separating the two or moremicroorganisms into individual isolates, selecting two or moreindividual isolates, and then combining the selected two or moreisolates.

In another embodiment, the method further comprises repeating steps a)to c) one or more times, wherein where two or more microorganisms areacquired in step c), the two or more microorganisms are separated intoindividual isolates, two or more individual isolates are selected andthen combined, and the combined isolates are used as the first set ofone or more microorganism in step a) of the successive repeat.Accordingly, where reference is made to using the one or moremicroorganisms acquired in step c) in step a) of the method, it shouldbe taken to include using the combined isolates of this embodiment ofthe disclosure.

In another embodiment, two or more methods of the disclosure may beperformed separately and the second set of one or more microorganismsacquired in step c) of each separate method combined. In one embodiment,the combined microorganisms are used as the first set of one or moremicroorganisms in step a) of any successive repeat of the method of thedisclosure.

In one embodiment, the methods of the first aspect of the disclosure mayalso be useful in identifying and/or selecting one or more endophyticmicroorganism capable of imparting one or more beneficial property to aplant.

In one embodiment, plant material (including for example seeds,seedlings, cuttings, and/or propagules thereof) may be used as thesource of microorganisms for step a). In a preferred embodiment, theplant material used as a source for microorganisms in step a) is seedmaterial. The plant material may be surface sterilised.

In another embodiment, the methods of the first aspect of the disclosuremay be useful in identifying and/or selecting one or more unculturablemicroorganism capable of imparting one or more beneficial property to aplant. In this embodiment, plant material (including for example seeds,seedlings, cuttings, and/or propagules thereof) may be used as thesource of microorganisms for step a). In a preferred embodiment, theplant material used as a source for microorganisms in step a) is explantmaterial (for example, plant cuttings). The plant material may besurface sterilised.

In a second broad aspect, there is provided a method for assisting inthe improvement of one or more plants according to a method as hereindescribed, comprising arranging for the evaluation of said plant(s) inthe presence of one or more microorganisms and/or compositions. Themethod preferably comprises at least the steps of a method of the first,seventh (and/or related) aspect, and/or the eighth (and/or related)aspect of the disclosure.

According to one embodiment, the plant(s) are for growing in a firstregion. The microorganism(s) may or may not (or at least to asignificant extent) be present in the first region.

“Region” and “first region” are to be interpreted broadly as meaning oneor more areas of land. The land areas may be defined bygeographical/political/private land boundaries or by land areas havingsimilar properties such as climate, soil properties, presence of aparticular pest, predominant plant community, predominant climate,predominant geological formation, etc.

In an aspect, the evaluation is performed in a second region in whichthe microorganism(s) are present. Microorganisms may be obtained fromother sources including microorganism depositaries and artificiallyassociated with plant material and/or soil. Furthermore, while plant(s)may be cultivated in essentially a conventional manner, but in a regionhaving microorganisms not normally associated with the plant(s), atleast in the first region, artificial growing environments mayalternatively be used as would be appreciated by those skilled in theart. Thus, possible beneficial microorganism/plant relationships may beidentified that would not necessarily normally be utilised.

Preferably, the step of arranging comprises arranging for one or moreof:

-   -   receipt or transmission of an identity of one or more plants or        plant types to be evaluated;    -   receipt or transmission of plant material from one or more        plants or plant types to be evaluated;    -   identification and/or selection of the microorganism(s) and/or        composition(s);    -   acquisition of the microorganism(s) and/or composition(s); and    -   associating the microorganism(s) and/or composition(s) with the        plant material.

Preferably, the method comprises evaluating (or arranging for saidevaluation of) said plant(s) in the presence of said microorganism(s)and/or composition(s).

The step of evaluating preferably comprises performing one or more ofthe steps of a method described herein, in particular embodiments amethod of one or more of the first aspect, seventh (and/or related)aspect or eighth (and/or related) aspect of the disclosure.

The various steps identified above may be performed by a single entityalthough at least two parties may be involved, a first party which makesa request and a second party which actions the request. Note thatvarious agents may act for one or both parties and that varying levelsof automation may be used. For example, in response to a particularrequest the microorganism(s) may be selected by a processor querying adatabase based on known microorganism associations for that or similarplant(s) with little or no input required from an operator.

Furthermore, the evaluation may be performed by the requesting partyand/or in the first region. Performing the evaluation in the firstregion better ensures that the evaluation is accurate and that nounforseen environmental factors that may impact on the plant(s) or themicroorganism(s) are not considered.

Following the evaluation or during the course thereof, the methodpreferably further comprises one or more of:

-   -   receiving or sending one or more microorganisms (or at least the        identity thereof) and/or composition(s) to the first region,        including in combination with plant material; and    -   growing said plant(s) or other plants (preferably having similar        properties) in the first region in the presence of said        microorganism(s) and/or composition(s).

The method of the second aspect may be embodied by a first party:

-   -   identifying a need for an improvement in a plant(s);    -   sending the identity thereof and/or relevant plant material to a        second party together with any relevant information, and    -   receiving plant material and/or one or more microorganisms        and/or the identities thereof and/or composition(s).

The step of receiving is preferably performed following or as a resultof an assessment of plant/microorganism and/or plant/compositionassociations. Preferably, the assessment is made using a method asdescribed herein, in particular embodiments a method of the firstaspect, the seventh (and/or related) aspect or the eighth (and/orrelated) aspect.

The method of the second aspect may additionally or alternatively beembodied by a second party:

-   -   receiving an identity of a plant(s) and/or relevant plant        material from a first party together with any relevant        information, and    -   sending plant material and/or one or more microorganism(s)        and/or the identities thereof and/or composition(s) to the first        party.

The step of sending is preferably performed following or as a result ofan assessment of plant/microorganism and/or plant/compositionassociations. Preferably, the assessment is made using a methoddescribed herein, in particular embodiments a method of the firstaspect, the seventh (and/or related) aspect or the eighth (and/orrelated) aspect.

According to a third aspect, there is provided a system for implementingthe method of the second aspect.

The system of the third aspect preferably includes one or more of:

-   -   means for receiving or transmitting an identity of one or more        plants or plant types to be evaluated;    -   means for receiving or transmitting plant material from one or        more plants or plant types to be evaluated;    -   means for identifying and/or selecting microorganism(s) and/or        composition(s);    -   means for acquiring the microorganism(s) and/or composition(s);    -   means for associating the microorganism(s) and/or composition(s)        with the plant material;    -   means for evaluating said plant(s) in the presence of said        microorganism(s) and/or composition(s);    -   means for receiving or sending one or more microorganisms (or at        least the identity thereof) and/or composition(s) to the first        region, including in combination with plant material; and    -   means for growing said plant(s) or other plants (preferably        having similar properties) in the first region in the presence        of said microorganism(s) and/or composition(s).

Means known to those skilled in the art may be used to provide thefunctionality required in the system of the third aspect. For example,conventional communication means, including the internet, may be used toconvey the identities of plants/microorganisms; conventional carriermeans may be used to convey the plantmaterial/microorganisms/composition(s); conventional means and processesmay be used to associate a microorganism and/or composition with plantmaterial and conventional means for evaluating said plant(s) and/or theplant/microorganism and/or plant/composition associations may be used.

According to an embodiment, the system of the disclosure is embodied bya facility configured to transmit request(s) for an improvement in aplant(s) and subsequently to receive plant material and/or one or moremicroorganisms and/or the identities thereof, preferably following or asa result of an assessment of plant/microorganism associations.Preferably, the assessment is made using a method described herein, inparticular embodiments a method of the first aspect, the seventh (and/orrelated) aspect, or the eighth (and/or related) aspect.

The system of the second aspect may additionally or alternatively beembodied by a facility configured to receive an identity of a plant(s)and/or relevant plant material from together with any relevantinformation; and send plant material and/or one or more microorganismsand/or the identities thereof and/or composition(s), preferablyfollowing or as a result of an assessment of plant/microorganism orplant/composition associations. Preferably, the assessment is made usinga method described herein, in particular embodiments a method of thefirst aspect, the seventh (and/or related) aspect or the eighth (and/orrelated) aspect.

Accordingly to a fourth broad aspect of the disclosure, there isprovided a microorganism acquired, selected, or isolated by a method asherein before described. In one embodiment, the microorganism is anendophyte. In one embodiment, the microorganism is unculturable.

In a fifth broad aspect of the disclosure, there is provided a methodfor the production of a composition to support plant growth, qualityand/or health or a composition to suppress or inhibit the growth,quality and/or health of a plant, the method comprising the steps of amethod herein before described and the additional step of combining theone or more microorganisms selected by the method with one or moreadditional ingredients.

In a sixth broad aspect of the disclosure, there is provided acomposition comprising one or more microorganism of the fourth broadaspect or as prepared by a method of the fifth broad aspect.

In a seventh broad aspect of the disclosure there is provided a methodfor the selection of a composition which is capable of imparting one ormore beneficial property to a plant, the method comprising at least thesteps of:

-   -   a) culturing one or more microorganisms selected by a method of        the first aspect of the disclosure in one or more media to        provide one or more culture;    -   b) separating the one or more microorganism from the one or more        media in the one or more culture after a period of time to        provide one or more composition substantially free of        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition of step b);    -   d) selecting one or more composition from step c) if it is        observed to impart one or more beneficial property to the one or        more plants.

In an aspect of the disclosure related to (but distinct from) theseventh broad aspect of the disclosure there is provided a method forthe selection of a composition which is capable of imparting one or morebeneficial property to a plant, the method comprising at least the stepsof:

-   -   a) culturing one or more microorganisms selected by a method of        the first aspect of the disclosure in one or more media to form        one or more culture;    -   b) inactivating the one or more culture of step a) to provide        one or more composition containing one or more inactivated        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition of step b);    -   d) selecting one or more composition from step c) if it is        observed to impart one or more beneficial property to the one or        more plants.

In an eighth broad aspect of the disclosure there is provided a methodfor the selection of one or more microorganisms, which are capable ofproducing a composition which is capable of imparting one or morebeneficial property to a plant, the method comprising at least the stepsof:

-   -   a) culturing one or more microorganism selected by a method of        the first aspect of the disclosure in one or more media to        provide one or more culture;    -   b) separating the one or more microorganism from the one or more        media in the one or more culture from step a) after a period of        time to provide one or more composition substantially free of        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition from step b);    -   d) selecting the one or more microorganisms associated with (or        in other words used to produce the) one or more composition        observed to impart one or more beneficial property to the one or        more plants.

In an aspect related to (but distinct from) the eighth broad aspect ofthe disclosure there is provided a method for the selection of one ormore microorganisms which are capable of producing a composition whichis capable of imparting one or more beneficial property to a plant, themethod comprising at least the steps of:

-   -   a) culturing one or more microorganism in one or more media to        provide one or more culture;    -   b) separating the one or more microorganism from the one or more        media in one or more culture after a period of time to provide        one or more composition substantially free of microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition of step b);    -   d) selecting the one or more microorganisms associated with (or        in other words used to produce the) one or more composition        observed to impart one or more beneficial property to the one or        more plants; and,    -   e) using the one or more microorganisms selected in step d) in        step a) of a method of the first, eighth or ninth aspects of the        disclosure.

In a related aspect, step b) of the method of the eighth (and/orrelated) aspect could be substituted with the step of b) inactivatingthe one or more culture of step a) to provide one or more compositioncontaining one or more inactivated microorganisms, and then using thiscomposition in step c) of the process.

It should be appreciated that the methods of the first, seventh (and/orrelated) and eighth (and/or related) aspects may be combined in anycombination, including the methods being run concurrently orsequentially in any number of iterations, with compositions and/ormicroorganisms selected or isolated from the methods being usedindividually or combined and used in iterative rounds of any one of themethods. By way of example, a method of the seventh (and/or related)aspect may be performed and a composition selected. The selection of acomposition indicates that the one or more microorganism separated fromthe media in step b) is desirable for imparting beneficial properties tothe one or more plant (as the one or more microorganism is capable ofproducing a selected composition). The one or more microorganism maythen be used in another round of a method of the first aspect, seventh(and/or related) aspect or eighth (and/or related) aspect.Alternatively, the combination of methods could be run in reverse. Thiscould be repeated any number of times in any order and combination.Accordingly the disclosure provides for the use of one or moremicroorganism, composition or plant acquired, selected or isolated by amethod of the disclosure in any other method of the disclosure.

In a ninth broad aspect of the disclosure there is provided acomposition obtained as a result of the methods of the seventh (and/orrelated) or eighth (and/or related) broad aspects of the disclosure.

In a tenth broad aspect of the disclosure there is provided acombination of two or more microorganisms acquired, selected, orisolated by a method as herein before described.

In another aspect, the disclosure provides the use of one or morecomposition and/or microorganism acquired, selected or isolated by amethod of the disclosure for imparting one or more beneficial propertyto one or more plant.

It should be appreciated that methods of the disclosure may also involveapplying steps a) to d) of the method of the first aspect on two or moredifferent species of plant so as to identify combinations ofmicroorganisms that may impart a positive benefit to one species and anegative benefit to another species simultaneously. For example, one maywish to identify a group of microorganisms that may simultaneouslyimprove the growth and survival of a food crop and suppress or inhibitthe growth of a competing crop or weed. This may be achieved by usingtwo or more different plant species in step a) or running separatemethods on different species and at appropriate points combining themicroorganisms acquired in those methods and conducting furtheriterations.

The disclosure also provides plants selected in a method of thedisclosure.

The disclosure also provides the use of a method of the disclosure in aplant breeding program, and a plant breeding program comprisingconducting a method of the disclosure.

In another aspect, the disclosure provides a composition comprising oneor more of the microorganisms listed in table 4. In one embodiment, theone or more microorganisms are endophytes.

In another aspect, the disclosure provides a composition comprising oneor more microorganisms listed in table 3. In some aspects, a microbialconsortium comprising at least one member chosen from the microbeslisted in table 3 and combinations thereof are provided.

In another aspect, the disclosure provides a composition comprising oneor more microorganisms listed in table 2. In some aspects, a microbialconsortium comprising at least one member chosen from the microbeslisted in table 2 and combinations thereof are provided.

In another aspect, the disclosure provides for the use of one or moremicroorganism listed in table 4 or a composition comprising same forincreasing plant biomass. In one embodiment, the plant is maize. In oneembodiment, the one or more microorganisms are endophytic. In someaspects, a microbial consortium comprising at least one member chosenfrom the microbes listed in table 4 and combinations thereof areprovided.

In another aspect, the disclosure provides for the use of one or moremicroorganisms listed in table 3 or a composition comprising same forincreasing carbohydrate concentrations in one or more plant. In oneembodiment, the one or more plant is basil.

In another aspect, the disclosure provides for the use of one or moremicroorganisms listed in table 2 or a composition comprising same forincreasing plant biomass. In one embodiment, the one or more plant isryegrass.

The disclosure may also be said broadly to consist in the parts,elements, and features referred to or indicated in the specification ofthe application, individually or collectively, in any or allcombinations of two or more of said parts, elements, or features, andwhere specific integers are mentioned herein which have knownequivalents in the art to which the disclosure relates, such knownequivalents are deemed to be incorporated herein as if individually setforth.

Further Embodiments of Accelerated Microbial Selection Methodology

Also described herein, is a method for selecting one or moremicroorganisms capable of imparting at least one beneficial property toa plant, comprising:

-   -   a) subjecting one or more plant to a growth medium in the        presence of a first set of one or more microorganisms;    -   b) selecting one or more plant following step a);    -   c) acquiring a second set of one or more microorganisms from        said one or more plant selected in step b);    -   d) repeating steps a) to c) one or more times, wherein the        second set of one or more microorganisms acquired in step c) is        used as the first set of microorganisms in step a) of any        successive repeat; and    -   e) selecting one or more microorganisms that is associated with        imparting a beneficial property to a plant.

Another embodiment taught herein, is a method for selecting one or moremicroorganisms capable of imparting at least one beneficial property toa plant, comprising:

-   -   a) subjecting one or more plant to a growth medium in the        presence of a first set of one or more microorganisms;    -   b) selecting one or more plant following step a);    -   c) acquiring a second set of one or more microorganisms from        said one or more plant selected in step b);    -   d) repeating steps a) to c) one or more times, wherein the        second set of one or more microorganisms acquired in step c) is        used as the first set of microorganisms in step a) of any        successive repeat; and    -   e) isolating one or more microorganisms associated with        imparting a beneficial property to a plant;    -   f) utilizing a molecular technique to characterize the one or        more microorganisms isolated in step e); and    -   g) selecting one or more characterized microorganisms that is        associated with imparting a beneficial property to a plant.

Further, the taught methods can include an additional step of h)combining the at least two microorganisms into a microbial consortium.In some instances, the microbes present at the end of the iterativeaccelerated microbial selection process will constitute a consortium. Inother instances, one will select microbes from one or more acceleratedmicrobial selection processes and then combine those microbes into aconsortium. The consortium can be constructed with individual microbesidentified in different accelerated microbial selection protocols. Forinstance, microbes identified in accelerated microbial selectionprotocols from previous years, geographic regions, plant species, soiltypes, etc, may be combined to construct a consortium.

In one embodiment, the one or more plant is ryegrass and the selectingof step b), of a second and any further successive repeat of stepsa)-c), is based upon selecting the ryegrass plants with the largestbiomass.

In another embodiment, the one or more plant is ryegrass and theselecting of step b), of a second and any further successive repeat ofsteps a)-c), is based upon selecting the ryegrass plants with thelargest biomass, and wherein the one or more microorganisms comprise amember selected from the group consisting of: Microbacteriumginsengiterrae, Bacillus cereus, Microbacterium oxydans, Rhizobiumpusense, Curtobacterium ginsengisoli, Penicillium daleae, Brevundimonasvesicularis, Aeromicrobium ponti, Microbacterium hydrocarbonoxydans,Sphingopyxis chilensis, Arthrobacter keyser, Penicillium melinii,Rhizobium grahamii, Brevundimonas vesicularis, Rhizobium pusense,Curtobacterium ginsengisoli, Herbaspirillum rubrisubalbicans, Rhizobiumetli, Exiguobacterium indicum, Mesorhizobium amorphae, Brevundimonasvesicularis, Arthrobacter keyser, and combinations thereof.

In yet another embodiment, the one or more plant is basil and theselecting of step b), of a second and any further successive repeat ofsteps a)-c), is based upon selecting the basil plants with the greatestmedian sugar content.

Further, another embodiment teaches that the one or more plant is basiland the selecting of step b), of a second and any further successiverepeat of steps a)-c), is based upon selecting the basil plants with thegreatest median sugar content, and wherein the one or moremicroorganisms comprise a member selected from the group consisting of:Sphingomonas mali, Flavobacterium micromati, Penicillium sp.,Sphingobium chlorophenolicum, Massilia niastensis, Flavobacteriumlimicola, Rhizobium alamii, Sphingopyxis sp., Pelomonas aquatica,Azospirillum hpoferum, Mesorhizobium amorphae, Asticcacaulis taihuensis,Ralstonia solanacearum, Microbacterium foliorum, Trichoderma,Burkholderia megapolitana, Mesorhizobium amorphae, Umbelopsis sp.,Aquabacterium fontiphilum, Rhodanobacter terrae, Sphingomonas mali,Sphingobium xenophagum, Pseudomonas moraviensis, Massilia niastensis,Flavobacterium limicola, Umbelopsis sp., and combinations thereof.

In another embodiment, the one or more plant is maize and the selectingof step b), of a second and any further successive repeat of stepsa)-c), is based upon selecting the maize plants with the largestbiomass.

Still further, an embodiment teaches that the one or more plant is maizeand the selecting of step b), of a second and any further successiverepeat of steps a)-c), is based upon selecting the maize plants with thelargest biomass, and wherein the one or more microorganisms comprise amember selected from the group consisting of: Herbaspirillumfrisingense, Acinetobacter sp., Xanthomonas translucens, Pseudomonasmarginalis, Herbiconiux ginsengi, Burkholderia cepacia, Microbacteriumoxydans, Pseudomonas moraviensis, Azotobacter chroococcum, Pseudomonasfrederiksbergensis, Sphingomonas rosa, Rhizobium endophyticum, Bacillusthioparans, Terriglobus roseus, Novosphingobium rosa, Azospirillumlipoferum, Streptomyces thermocarboxydus, Herbaspirillum frisingense,and combinations thereof.

The disclosure also presents a method of creating a microbial consortiumcapable of promoting at least one beneficial plant phenotypic trait,comprising:

-   -   a) subjecting at least one plant to a growth medium in the        presence of a first plurality of microorganisms;    -   b) selecting at least one plant following step a);    -   c) acquiring a second plurality of microorganisms from said at        least one plant selected in step b);    -   d) repeating steps a) to c) one or more times, wherein the        second plurality of microorganisms acquired in step c) is used        as the first plurality of microorganisms in step a) of any        successive repeat;    -   e) isolating at least two microorganisms from said plurality of        microorganisms that are associated with promoting at least one        beneficial plant phenotypic trait;    -   f) utilizing a molecular technique to characterize the at least        two isolated microorganisms;    -   g) selecting the at least two characterized microorganisms; and    -   h) combining the at least two microorganisms into a microbial        consortia

In a particular embodiment, the characterization of step f) comprises:determining the relative abundance of the at least two microorganismsthat are associated with promoting at least one beneficial plantphenotypic trait.

In another embodiment, the characterization of step f) comprises:determining the relative abundance of the at least two microorganismsthat are associated with promoting at least one beneficial plantphenotypic trait; and wherein the selecting of step g), comprises:choosing the at least two characterized microorganisms based on anincrease in their relative abundance compared to their abundance from aprevious iteration of steps a)-f). Also, the disclosure teaches anembodiment, wherein the at least two microorganisms chosen comprisemicroorganisms whose relative abundance increased at least 100%.

In an embodiment, the methods increase the frequency of the bestmicrobes for a specific desired plant phenotype through iterativeselection (selection on a plant phenotypic trait) and then the bestconsortia of microbes are isolated and applied as a seed or soiltreatment. The disclosed methods are more powerful and efficient thantraditional methods based upon mixing alreadyknown/characterized/identified microbes to create a microbialconsortium. In embodiments, the present methods are not dependent uponthe a priori existence of a known and characterized microbe. Rather, theiterative selection methods, in embodiments, are able to identify thebest microbes for promoting a plant phenotypic trait, and only then arethe microbes identified, if so desired.

In aspects, the directed evolution of a microbial consortium involvesiterative selection of the best performing plants based upon the plantsexpression of a target trait and then subsequently transmitting theevolving microbial consortium to the next generation of plants throughthe iterative selection process. The iterative selection process can berepeated until the desired plant phenotype is achieved.

In an embodiment, directed evolution of a microbial community involves:

-   -   a) Populating the initial plant growth environment (soil or        medium);    -   b) Growing plants without pressure;    -   c) Selecting best-performing individual plants (and the        associated microbes);    -   d) Recovering microbes from roots and foliage of selected        plants;    -   e) Using these microbes to populate the soil/medium for the next        round of plant growth and selection (many methods);    -   f) Repeating iterative selection process 2-5 (on average, can be        any number) times until the improvement of the desired trait is        achieved or plateaus;    -   g) After the terminal iteration, then isolating the microbes        from the best-performing plants;    -   h) Reconstructing the microbial associations and developing a        microbial consortia product.

The disclosed methods allow for the microbial consortia to be used withany other plant trait. Also, the methods taught herein enable improvedfertilizer efficiency to minimize cost and environmental impact, allowfor the production of more food per acre, and promote the production ofmore biomass for biofuels or lock up carbon.

In embodiments, methods taught herein use a multitude of moleculartechniques, including: community fingerprinting—ARISA (AutomatedRibosomal Intergenic Spacer Analysis); microbiome analysis to identifyorganisms via high throughput DNA sequencing & phylogenic analysis;microbial isolation & characterization via high throughput functionalassays & improved culture techniques; quantitative analysis & microbetracking via quantitative real time PCR (qRT-PCR) or fluorescent in situhybridization (FISH) microscopy.

The taught methods allow for the monitoring of plant gene expressionthroughout the iterative process such that one can infer relationshipsbetween changes in the microbiome, plant gene expression, and the planttrait.

Thus, in embodiments, comparative analysis will enable one to identifynovel plant genes and pathways that may serve as targets for cropimprovement through: traditional breeding; plant genetic engineering;and targets for chemical induction or repression (i.e. drugabletargets).

In an embodiment, the outcome of the methodology taught herein ismicrobes that improve targeted crop traits such as nutrient useefficiency, biological and physical challenges, as well as microbes thancan assist crop breeding programs and improve the effectiveness anddurability of transgenic crop traits.

In certain aspects, the accelerated microbial selection methodologycreates multiple beneficial traits by using microbes (or consortia) withdifferent beneficial effects in a single crop. Thus, the disclosedmethods allow for the engineering of crop-microbe interactions viaunderstanding of crop trait gene expression patterns and construction ofrecombinant microbes engineered to consolidate optimized activity in asingle organism.

Definitions

Numbers and numerical ranges recited herein are to be understood to bemodified by the term “about” as would be understood by one of ordinaryskill in the art.

“About” can mean plus or minus a percent (e.g., ±5%) of the number,parameter, or characteristic so qualified, which would be understood asappropriate by a skilled artisan to the scientific context in which theterm is utilized. Furthermore, since all numbers, values, andexpressions referring to quantities used herein, are subject to thevarious uncertainties of measurement encountered in the art, then unlessotherwise indicated, all presented values may be understood as modifiedby the term “about.”

As used herein, the articles “a,” “an,” and “the” may include pluralreferents unless otherwise expressly limited to one-referent, or if itwould be obvious to a skilled artisan from the context of the sentencethat the article referred to a singular referent.

Where a numerical range is disclosed herein, then such a range iscontinuous, inclusive of both the minimum and maximum values of therange, as well as every value between such minimum and maximum values.

Still further, where a range refers to integers, every integer betweenthe minimum and maximum values of such range is included. In addition,where multiple ranges are provided to describe a feature orcharacteristic, such ranges can be combined. That is to say that, unlessotherwise indicated, all ranges disclosed herein are to be understood toencompass any and all subranges subsumed therein. For example, a statedrange of from “1 to 10” should be considered to include any and allsubranges between the minimum value of 1 and the maximum value of 10.Exemplary subranges of the range “1 to 10” include, but are not limitedto, 1 to 6.1, 3.5 to 7.8, and 5.5 to 10.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms, used interchangeably, include but are not limitedto the two prokaryotic domains, Bacteria and Archaea, as well aseukaryotic fungi and protists.

The term “microbial consortia” refers to a subset of a microbialcommunity of individual microbial species or strains of a species thatcan be described as carrying out a common function, or can be describedas participating in, or leading to, or correlating with, a recognizableparameter or plant phenotypic trait. The community may comprise two ormore species or strains of a species of microbes. In some instances, themicrobes coexist within the community symbiotically.

The term “microbial community” means a group of microbes comprising twoor more species or strains.

The term “directed evolution” is used in the broadest sense of the word“evolve” and does not necessarily refer to Mendelian inheritance. Thus,to “evolve” means to change. This change can be brought about by variousparameters. In the examples that follow, a microbial community isevolved, i.e. the microbial community changes, over iterative selectionsteps according to the taught methods. In some embodiments, afterseveral iterative rounds of accelerated microbial selection, themicrobial community that results is drastically different from themicrobial community present at the start of the method. Thus, in someembodiments, the methods take a random and heterogeneous microbialcommunity, said members not necessarily working toward a desiredfunction, but over the course of the iterative selection steps of thetaught methods, a microbial community begins to emerge, whereinmicrobial species participate/correlate to a desired function, e.g.increasing a plant phenotypic trait of interest.

The term “accelerated microbial selection” or “AMS” is usedinterchangeably with the term “directed microbial selection” or “DMS”and refers to the iterative selection methodology elaborated upon in thedisclosure.

The disclosure utilizes the term “plant” broadly to include all plantparts, seeds, seedlings, cuttings, propagules, root, stem, and/or foliartissue. Further, a plant may be defined as the intimately associatedplant rhizosphere.

A plant rhizosphere may include any component of the growth mediainfluenced by the plant and its associated microbiome.

It should be appreciated that as referred to herein a “beneficialproperty to a plant” should be interpreted broadly to mean any propertywhich is beneficial for any particular purpose including propertieswhich may be beneficial to human beings, other animals, the environment,a habitat, an ecosystem, the economy, of commercial benefit, or of anyother benefit to any entity or system. Thus, the benefit could be adesired change to a soil or growth medium. Accordingly, the term shouldbe taken to include properties which may suppress, decrease, or blockone or more characteristic of a plant, including suppressing,decreasing, or inhibiting the growth or growth rate of a plant. Thedisclosure may be described herein, by way of example only, in terms ofidentifying positive benefits to one or more plants or improving plants.However, it should be appreciated that the disclosure is equallyapplicable to identifying negative benefits that can be conferred toplants. Such beneficial properties include, but are not limited to, forexample: improved growth, health and/or survival characteristics,suitability or quality of the plant for a particular purpose, structure,colour, chemical composition or profile, taste, smell, improved quality.In other embodiments, beneficial properties include, but are not limitedto, for example: decreasing, suppressing or inhibiting the growth of aplant identified to be a weed; constraining the height and width of aplant to a desirable ornamental size; limiting the height of plants usedin ground cover applications such as motorway and roadside banks anderosion control projects; slowing the growth of plants used in turfapplications such as lawns, bowling greens and golf courses to reducethe necessity of mowing; reducing ratio of foliage/flowers in ornamentalflowering shrubs; regulate production of and/or response to plantpheromones (resulting in increased tannin production in surroundingplant community and decreased appeal to foraging species).

As used herein, “improved” should be taken broadly to encompassimprovement of a characteristic of a plant which may already exist in aplant or plants prior to application of the disclosure, or the presenceof a characteristic which did not exist in a plant or plants prior toapplication of the disclosure. By way of example, “improved” growthshould be taken to include growth of a plant where the plant was notpreviously known to grow under the relevant conditions.

As used herein, “inhibiting and suppressing” and like terms should betaken broadly and should not be construed to require complete inhibitionor suppression, although this may be desired in some embodiments.

To assist in describing the disclosure, the terms a “first set of one ormore microorganisms” and a “second set of one or more microorganisms”may be used herein to distinguish the set or group of microorganism(s)applied in step a) and the set or group of microorganism(s) acquired instep c) of a method of the disclosure. In certain embodiments, the setsof microorganisms will be distinct; for example, the second set may be asubset of the first set, as a result of combining the first set with theplant and then selecting one or more plant based on one or moreselection criterion. However, it should be appreciated that this may notalways be the case and accordingly, the use of this terminology shouldnot be construed in such a limited manner.

As is further described herein, microorganism(s) may be contained withina plant, on a plant, and/or within the plant rhizosphere or the plantgrowth medium. Accordingly, where reference is made herein to acquiringa second set of one or more microorganisms “from” a plant, unless thecontext requires otherwise, it should be taken to include reference toacquiring a second set of microorganisms contained within a plant, on aplant and/or within the plant rhizosphere, or also from the plant growthmedium.

For ease of reference, the wording “associated with” may be usedsynonymously to refer to microorganism(s) contained within a plant, on aplant and/or within the plant rhizosphere.

As used herein, “isolate”, “isolated” and like terms should be takenbroadly. These terms are intended to mean that the one or moremicroorganism(s) has been separated at least partially from at least oneof the materials with which it is associated in a particular environment(for example soil, water, plant tissue). “Isolate”, “isolated” and liketerms should not be taken to indicate the extent to which themicroorganism(s) has been purified.

As used herein, “individual isolates” should be taken to mean acomposition or culture comprising a predominance of a single genera,species or strain of microorganism, following separation from one ormore other microorganisms. The phrase should not be taken to indicatethe extent to which the microorganism has been isolated or purified.However, “individual isolates” preferably comprise substantially onlyone genus, species or strain of microorganism.

The term “growth medium” as used herein, should be taken broadly to meanany medium which is suitable to support growth of a plant. By way ofexample, the media may be natural or artificial including, but notlimited to, soil, potting mixes, bark, vermiculite, hydroponic solutionsalone and applied to solid plant support systems, and tissue culturegels. It should be appreciated that the media may be used alone or incombination with one or more other media. It may also be used with orwithout the addition of exogenous nutrients and physical support systemsfor roots and foliage.

A “composition to support plant growth, health, and/or quality” shouldbe taken broadly to include compositions which may assist the growth,general health and/or survival of a plant, the condition of a plant, orassist in the maintaining or promoting any desired characteristic,quality, and/or trait. It should be taken to include maintaining oraltering the production of one or more metabolite or other compound by aplant as well altering gene expression and the like. The phrase shouldnot be taken to imply that the composition is able to support plantgrowth, quality and/or health on its own. However, in one embodiment thecompositions are suitable for this purpose. Exemplary compositions ofthis aspect of the disclosure include but are not limited to plantgrowth media, plant mineral supplements and micronutrients, composts,fertilisers, potting mixes, insecticides, fungicides, media to protectagainst infection or infestation of pests and diseases, tissue culturemedia, seed coatings, hydroponic media, compositions that imparttolerance to drought or abiotic stress such as metal toxicity,compositions that modify soil pH.

A “composition to inhibit or suppress plant growth, health, and/orquality” should be taken broadly to include compositions which mayassist in suppressing or inhibiting one or more characteristic, qualityand/or trait of a plant, including its growth, general health and/orsurvival. It should be taken to include maintaining or altering theproduction of one or more metabolite or other compounds by a plant aswell altering gene expression and the like. The phrase should not betaken to imply that the composition is able to suppress or inhibit plantgrowth, quality and/or health on its own. However, in one embodiment thecompositions are suitable for this purpose. Exemplary compositions ofthis aspect of the disclosure include but are not limited to plantgrowth suppression media, weed killer, fertilisers, potting mixes, plantmineral supplements and micronutrients, composts, mixes, insecticides,fungicides, tissue culture media, seed coatings, hydroponic media,compositions that impart tolerance to drought or abiotic stress such asmetal toxicity, and compositions that modify soil pH.

As used herein “inactivating” the one or more culture and “inactivatedmicroorganisms” and like terms should be taken broadly to mean that themicroorganisms are substantially inactivated, fixed, killed or otherwisedestroyed. The term should not be taken to mean that all microorganismsare inactivated, killed or destroyed, however, this may be preferable.In one embodiment, the microorganisms are inactivated, fixed, killed ordestroyed to the extent that self-sustained replication is no longermeasurable using techniques known to one skilled in the art.

As used herein, a “composition substantially free of microorganisms”should be taken broadly and not be construed to mean that nomicroorganisms are present, although this may be preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present disclosure will become apparentfrom the following description, which is given by way of example only,with reference to the accompanying figures, in which:

FIG. 1: shows a system according to an embodiment of the disclosure.

FIG. 2: shows the process flow of a method of an embodiment of thedisclosure.

FIG. 3: shows a generalized process schematic of a disclosed method ofaccelerated microbial selection, also referred to herein as directedmicrobial selection. When the process is viewed in the context of amicrobial community, the schematic is illustrative of a process ofdirected evolution of a microbial community.

FIG. 4: shows a generalized process flow chart of an embodiment of thetaught methods.

FIG. 5: shows a graphic representation and associated flow chart of anembodiment of the disclosed methods.

FIG. 6 shows a graphic representation and associated flow chart of anembodiment of the disclosed methods. The figure illustrates the abilityto evolve microbial communities and selection of consortia for impartinga desirable phenotypic trait in a plant.

FIG. 7: shows a graphic representation and associated flow chart of anembodiment of the disclosed methods and illustrates that the methods canutilize microbes from a variety of sources (including multiple locationsfrom a single plant) and can select microbes that help develop a myriadof plant phenotypic traits.

FIG. 8: shows a process of microbial community (microbiome) analysisutilizing community fingerprinting (ARISA)+NextGen Sequencing.

FIG. 9: shows ARISA microbial community fingerprinting of the microbialcommunities in 5 different soils. The operational taxonomic units (OTU)identified in each soil by NextGen sequencing are given.

FIG. 10: shows ARISA microbial community fingerprinting of a soil sampleand the rhizosphere community of a corn seedling grown in the same soil.The figure illustrates that a plant (i.e. corn) can amplify microbespresent in its surroundings.

FIG. 11: shows ARISA microbial community fingerprinting across 5 soiltypes that have corn plants grown therein. The figure illustrates that aplant (i.e. corn) can “capture” different microbes depending upon thesoil it is growing within.

FIG. 12: shows the degree of similarity of the microbial communitiesassociated with 5 soil types, and with or without the same variety ofcorn growing in each of the soils. The graphic illustrates that themicrobial communities associated with the corn plants are distinct fromthe microbial communities with no corn across all 5 soil types. Thus,the methods disclosed herein are able to select for distinct microbialpopulations by utilizing a step of selecting for particular plants.

FIG. 13: shows the degree of similarity of the microbial communitiesassociated with multiple crop types all grown within the same soil type.The graphic also shows the microbial communities associated with thesingle soil type with no crops grown therein. The graphic illustratesthat the microbial communities associated with soils with no plantsgrown therein are different from those with plants growing therein.Further, each plant type can be seen to “capture” a particular communityof microbes.

FIG. 14: shows NextGen Sequencing microbial community data thatillustrates that the corn microbiome has changed after one round ofaccelerated microbial selection, also termed directed selection ofmicrobes “DSM”. The graphic shows the starting soil microbial community,the microbial community after “capture”, and the microbial communityafter one round of selection (R1) according to the taught methods.Colored squares=communities identified in soil samples. Each colorrepresents a different soil sample. Red symbols correspond to soilsample 5, as indicated in the experiment design graphic (i.e. FIG. 8).Triangles=rhizosphere communities identified in samples from microbecapture. Circles=rhizosphere communities from round 1 plants grown inSoil 5. Diamond=rhizosphere communities from round 1 plants grown insterile sand & vermiculite. The MDS plot is constructed from data of theexperiment from FIG. 8.

FIG. 15: shows that the methods of the disclosure drive dynamic changein relevant microbial abundance. The columns illustrate microbialabundance for the natural soil, microbial abundance after capture, andmicrobial abundance after Round 1 of a method as taught (R1). As can beseen, a microbial species that is normally ubiquitous in the soil“Species 21” is brought to below a detectable abundance level after R1.Also, “Species 205” that was not at a detectable abundance level in thenatural soil is brought to an abundance of 1642 after the capture stepand an abundance of 2487 after R1. Further, the methods also brought“Species 196” up from below detectable limits in natural soil to 131after R1.

FIG. 16: shows that the methods of the disclosure drive dynamic changein relevant microbial abundance. For example, “Species 205” was not at adetectable abundance level in the natural soil, but the taught methodsbring its abundance to 1642 after the capture step and an abundance of2487 after R1. These data enable targeted isolation and selection ofcomponents of the evolved community for the construction of microbialconsortia that can be linked to trait improvement by changes in relativeabundance.

FIG. 17: is an overview of data obtained by utilizing the taught methodsacross a variety of plants: corn, ryegrass, and basil; and a variety ofphenotypic plant traits: growth via endophytes in corn, growth inryegrass, and ability to increase sugar content in basil.

FIG. 18: is an overview of an experimental protocol implementing anaccelerated method of microbial selection to improve the growth of aplant in 9 months.

DETAILED DESCRIPTION

The following is a description of embodiments of the present disclosure.The disclosure will be further elucidated from the Examples providedhereafter.

The inventor(s) have found that one can readily identify microorganismscapable of imparting one or more beneficial property to one or moreplants through use of a method of the disclosure. The method is broadlybased on the presence of variability (such as genetic variability, orvariability in the phenotype for example) in the plants and microbialpopulations used. The inventors have identified that this variabilitycan be used to support a directed process of selection of one or moremicroorganisms, of use to a plant, and for identifying particularplant/microbe combinations which are of benefit for a particularpurpose, and which may never have been recognised using conventionaltechniques.

Consequently, because the methods disclosed herein present anaccelerated method of microbial selection, the methods are alsoapplicable to a method of directed evolution of communities of microbes.That is, particular individual microbes and communities of microbes thatare selected for by the accelerated selection methods lead to theevolution and development of the best consortia of microbes forproducing a phenotypic plant trait of interest.

The methods of the disclosure may be used as a part of a plant breedingprogram. The methods may allow for, or at least assist with, theselection of plants which have a particular genotype/phenotype which isinfluenced by the microbial flora, in addition to identifyingmicroorganisms and/or compositions that are capable of imparting one ormore property to one or more plants.

In one aspect, the disclosure relates to a method for the selection ofone or more microorganism(s) which are capable of imparting one or morebeneficial property to a plant.

Broadly, the method comprises in one aspect, at least the steps of a)growing one or more plant in a growth medium in the presence of a firstset of one or more microorganisms; b) selecting one or more plantfollowing step a); and, c) acquiring a second set of one or moremicroorganisms associated with said one or more plant selected in stepb). The one or more plants, growth medium and one or more microorganismsmay be provided separately and combined in any appropriate order priorto step a). In particular, the disclosure provides an iterative methodin which steps a) to c) may be repeated one or more times, wherein theone or more microorganisms acquired in step c) are used in step a) ofthe next cycle of the method. In one embodiment, steps a) to c) arerepeated once. In another embodiment, steps a) to c) are repeated twice.In another embodiment, steps a) to c) are repeated three times. Inanother embodiment, steps a) to c) are repeated at least until a desiredbeneficial property is observed.

It will be appreciated that after a desired number of repeats of stepsa) to c) the method may conclude with the acquisition of a set of one ormore microorganisms from step c).

The set of microorganisms acquired during the iterative process of stepsa) to c) can be a consortium of microbes that work together toward acommon function or correlate with a function. Often, that commonfunction relates to the development of a particular plant phenotypictrait of interest. By iteratively performing steps a)-c) the microbialcommunity can evolve to include the most appropriate members of thecommunity that correlate with a plant phenotypic trait of interest.

It should be appreciated that the methods do not require theidentification of the microorganisms in the population acquired in stepc) nor do they require a determination of the properties of individualmicroorganisms or combinations of microorganisms acquired. However,evaluation, identification, and/or a determination of the beneficialproperties could be conducted if desired. For example, it may bepreferred in some cases to isolate and identify the microbes in thefinal step of a method of the disclosure to determine their safety forcommercial use and to satisfy regulatory requirements. In such cases,genetic and/or phenotypic analyses may be conducted.

Further, when developing a community, or consortia of microbes thatcooperate towards, or correlate with, a particular function, it may bebeneficial to know the identity of such consortia members, though thisis not required.

In one embodiment, step a) is conducted using at least two plants. Inother embodiments 10 to 20 plants are used. In yet other embodiments, 20or more, 50 or more, 100 or more, 300 or more, 500 or more, or 1000 ormore plants are used.

As noted hereinbefore, where two or more plants are used in a particularmethod of the disclosure they need not be the same variety or species.

For example, in one embodiment it may be desirable to selectmicroorganisms that can impart a positive benefit to one plant varietyor species and a negative benefit to another plant variety or species.

For example, the disclosed methods can be utilized to encourage thedevelopment of a desired phenotypic trait in a commercially importantagriculture species while simultaneously imparting a negative influenceon an undesirable weedy species that is often associated with thecultivation of the agricultural crop.

In one embodiment, where two or more microorganisms are acquired in stepc), the method may further comprise the steps of separating the two ormore microorganisms into individual isolates, selecting two or moreindividual isolates, and then combining the selected two or moreisolates. This may result in the set of microorganisms acquired at theconclusion of a method of a disclosure.

However, in one embodiment, the combined isolates may then be used instep a) of successive rounds of the method. By way of example, from two,three, four, five, six, seven, eight, nine, ten, or more individualisolates may be combined. The inventors envisage an iterative method inwhich steps a) to c) are repeated one or more times, utilising theseadditional steps of separating, selecting, and combining with eachrepeat of the method, or interspersed or otherwise combined with amethod in which individual isolates are not selected and combined.

In an embodiment, this procedure describes a process of evolving themicrobial community, in the sense that the community (viewed as aplurality or community of microbes) changes and develops in response tothe iterative accelerated selection procedure.

It is expected that these combinations will detect previously unknown,desirable property promoting (such as plant growth), synergisticinteractions between microbes.

Using the iterative steps a) to c) will drive the starting population oftwo or more microorganisms toward microbes that interact with the plantto impart a desired property or characteristic. In other words, theprocess will allow for enrichment of suitable microorganisms within theplant microbiome.

As aforementioned, another way to characterize the iterative acceleratedselection process is by viewing the process based upon the evolvingcommunity of microbes or consortia that work together toward a commonfunction. Often, these microbial consortia will be correlated withhelping to promote and develop a desired plant characteristic orphenotypic trait. Consequently, the disclosed methods present a processof directed evolution of microbial consortia. In aspects, this is aunique method by which to direct the evolution of microbial consortia,as the microbes do not have to be associated with one another naturallynor do the microbes have to ever have been naturally exposed to theplant or substrate utilized in the iterative selection process.Therefore, the directed evolution of microbial consortia achievedutilizing the present methods allows for, in some embodiments, thedevelopment of completely synthetic consortia that are highlyspecialized for working together to promote a plant phenotypic trait ina plant that the microbes would not naturally encounter.

Selection of individual isolates may occur on the basis of anyappropriate selection criteria. For example, it may be random, it may bebased on the beneficial property or properties observed by performing amethod of the disclosure or, where information about the identity of themicroorganism is known, it may be on the basis that the microorganismhas previously been recognised to have a particular beneficial property.

In addition, two or more methods of the disclosure may be performedseparately or in parallel and the microorganisms that result from eachmethod combined into a single composition. For example, two separatemethods may be performed, one to identify microorganisms capable ofimparting one or more first beneficial property, and a second toidentify microorganisms capable of imparting one or more secondbeneficial property. The separate methods may be directed to identifyingmicroorganisms having the same beneficial property or having distinctbeneficial properties. The microorganisms and plants used in theseparate methods may be the same or different. If further optimisationof the microorganisms is desired, the single composition ofmicroorganisms may be applied to one or more further rounds of a methodof the disclosure. Alternatively, the single composition ofmicroorganisms may be used, as desired, to confer the relevantproperties to plant crops, without further optimisation. Combining twoor more methods of the disclosure in this way allows for the selectionand combination of microorganisms which may ordinarily be separated bytime and/or space in a particular environment.

In certain embodiments of the disclosure, the methods may comprisegrowing or propagating one or more plants selected in step c) of themethod, to grow the population of the second set of one or moremicroorganisms associated with the selected one or more plants, eitherat the conclusion of a method of the disclosure, or prior to using thesecond set of one or more microorganisms in step a) of any successiverepeat of the method. If the one or more plants (with associatedmicroorganisms) are grown or propagated at the conclusion of a method ofthe disclosure they may then be used or sold in that form.

Alternatively, one or more microorganisms may be isolated from the oneor more plants, or one or more plant tissue and/or one or more plantpart with associated microorganisms may be used as a crude source of theone or more microorganisms in any successive repeat of the disclosure,or for any other purpose at the conclusion of the method. In oneembodiment, the seeds (with associated microorganisms) of one or moreplant that has been grown or propagated may be obtained and used as asource of the one or more microorganisms in any successive repeat of themethod. Alternatively, if obtained at the conclusion of a method of thedisclosure, the seeds and associated microorganisms may be sold or usedfor any other purpose.

Further, the microbial community that has been evolved to impart and/orencourage the development of a plant phenotypic trait of interest may besold or used for any purpose. In particular embodiments, the individualmicrobes, microbial consortia, or microbial community, derived by thepresent methods can be formulated as a composition that is utilized as aseed coating for commercially important agricultural crops.

Further, the individual microbes, or microbial consortia, or microbialcommunities, developed according to the disclosed methods can becombined with known actives available in the agricultural space, suchas: pesticide, herbicide, bactericide, fungicide, insecticide, virucide,miticide, nemataicide, acaricide, plant growth regulator, rodenticide,anti-algae agent, biocontrol or beneficial agent. Further, the microbes,microbial consortia, or microbial communities developed according to thedisclosed methods can be combined with known fertilizers. Suchcombinations may exhibit synergistic properties.

In some embodiments, when the microbe or microbial consortia identifiedaccording to the taught methods is combined with an active chemicalagent one witnesses an additive effect on a plant phenotypic trait ofinterest.

In some embodiments, when the microbe or microbial consortia identifiedaccording to the taught methods is combined with a fertilizer onewitnesses an additive effect on a plant phenotypic trait of interest.

In other embodiments, when the microbe or microbial consortia identifiedaccording to the taught methods is combined with an active chemicalagent one witnesses a synergistic effect. The synergistic effectobtained by the taught methods can be quantified according to Colby'sformula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S., “CalculatingSynergistic and Antagonistic Responses of Herbicide Combinations”, 1967Weeds, vol. 15, pp. 20-22, incorporated herein by reference in itsentirety. Thus, by “synergistic” is intended a component which, byvirtue of its presence, increases the desired effect by more than anadditive amount. The microbes and consortia of the present methods cansynergistically increase the effectiveness of agricultural activecompounds and also agricultural auxiliary compounds.

In other embodiments, when the microbe or microbial consortia identifiedaccording to the taught methods is combined with a fertilizer onewitnesses a synergistic effect.

The composition comprising a microbial consortia developed according tothe disclosure can be formulated with certain auxiliaries in order toimprove the activity of a known active agricultural compound. This hasthe advantage that the amounts of active ingredient in the formulationmay be reduced while maintaining the efficacy of the active compound,thus allowing costs to be kept as low as possible and any officialregulations to be followed. In individual cases, it may also possible towiden the spectrum of action of the active compound since plants, wherethe treatment with a particular active ingredient without addition wasinsufficiently successful, can indeed be treated successfully by theaddition of certain auxiliaries along with the disclosed microbialconsortia. Moreover, the performance of the active may be increased inindividual cases by a suitable formulation when the environmentalconditions are not favorable.

Such auxiliaries that can be used in a composition comprising an activeagricultural compound and a microbial consortia developed according tothe disclosed methods can be an adjuvant. Frequently, adjuvants take theform of surface-active or salt-like compounds. Depending on their modeof action, they can roughly be classified as modifiers, activators,fertilizers, pH buffers, and the like. Modifiers affect the wetting,sticking, and spreading properties of a formulation. Activators break upthe waxy cuticle of the plant and improve the penetration of the activeingredient into the cuticle, both short-term (over minutes) andlong-term (over hours). Fertilizers such as ammonium sulfate, ammoniumnitrate or urea improve the absorption and solubility of the activeingredient and may reduce the antagonistic behavior of activeingredients. pH buffers are conventionally used for bringing theformulation to an optimal pH.

Further methods and aspects of the disclosure are described hereinafter.

Microorganisms

As used herein the term “microorganism” should be taken broadly. Itincludes but is not limited to the two prokaryotic domains, Bacteria andArchaea, as well as eukaryotic fungi and protists.

By way of example, the microorganisms may include Proteobacteria (suchas Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium,Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium,Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium andHalomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus,Mycoplasma, and Acetobacterium), Actinobacteria (such as Streptomyces,Rhodococcus, Microbacterium, and Curtobacterium), and the fungiAscomycota (such as Trichoderma, Ampelomyces, Coniothyrium,Paecoelomyces, Penicillium, Cladosporium, Hypocrea, Beauveria,Metarhizium, Verticullium, Cordyceps, Pichea, and Candida, Basidiomycota(such as Coprinus, Corticium, and Agaricus) and Oomycota (such asPythium, Mucor, and Mortierella).

In one embodiment, the microorganism is an endophyte or an epiphyte or amicroorganism inhabiting the plant rhizosphere. In one embodiment, themicroorganism is a seed-borne endophyte.

In certain embodiments, the microorganism is unculturable. This shouldbe taken to mean that the microorganism is not known to be culturable oris difficult to culture using methods known to one skilled in the art.

Microorganisms of use in the methods of the present disclosure (forexample, the first set of one or more microorganisms) may be collectedor obtained from any source or contained within and/or associated withmaterial collected from any source. In some embodiments, themicroorganisms are collected, obtained, captured, or otherwise derived,from a soil media (or any growth media). In some embodiments, themicroorganisms are collected, obtained, captured, or otherwise derived,from a soil media (or any growth media) and are not otherwise associatedwith a particular plant.

In one embodiment, the first set of one or more microorganisms areobtained from any general terrestrial environment, including its soils,plants, fungi, animals (including invertebrates) and other biota,including the sediments, water and biota of lakes and rivers; from themarine environment, its biota and sediments (for example sea water,marine muds, marine plants, marine invertebrates (for example sponges),marine vertebrates (for example, fish)); the terrestrial and marinegeosphere (regolith and rock, for example crushed subterranean rocks,sand and clays); the cryosphere and its meltwater; the atmosphere (forexample, filtered aerial dusts, cloud and rain droplets); urban,industrial and other man-made environments (for example, accumulatedorganic and mineral matter on concrete, roadside gutters, roof surfaces,road surfaces).

In another embodiment the first set of one or more microorganisms areobtained from a source likely to favour the selection of appropriatemicroorganisms. By way of example, the source may be a particularenvironment in which it is desirable for other plants to grow, or whichis thought to be associated with terroir. In another example, the sourcemay be a plant having one or more desirable traits, for example a plantwhich naturally grows in a particular environment or under certainconditions of interest. By way of example, a certain plant may naturallygrow in sandy soil or sand of high salinity, or under extremetemperatures, or with little water, or it may be resistant to certainpests or disease present in the environment, and it may be desirable fora commercial crop to be grown in such conditions, particularly if theyare, for example, the only conditions available in a particulargeographic location. By way of further example, the microorganisms maybe collected from commercial crops grown in such environments, or morespecifically from individual crop plants best displaying a trait ofinterest amongst a crop grown in any specific environment: for examplethe fastest-growing plants amongst a crop grown in saline-limitingsoils, or the least damaged plants in crops exposed to severe insectdamage or disease epidemic, or plants having desired quantities ofcertain metabolites and other compounds, including fibre content, oilcontent, and the like, or plants displaying desirable colours, taste orsmell. The microorganisms may be collected from a plant of interest orany material occurring in the environment of interest, including fungiand other animal and plant biota, soil, water, sediments, and otherelements of the environment as referred to previously.

In certain embodiments, the microorganisms are sourced from previouslyperformed methods of the disclosure (for example, the microorganismsacquired in step c) of the method), including combinations of individualisolates separated from the second set of microorganisms isolated instep c) or combinations of microorganisms resulting from two or moreseparately performed methods of the disclosure.

While the disclosure obviates the need for pre-existing knowledge abouta microorganism's desirable properties with respect to a particularplant species, in one embodiment a microorganism or a combination ofmicroorganisms of use in the methods of the disclosure may be selectedfrom a pre-existing collection of individual microbial species orstrains based on some knowledge of their likely or predicted benefit toa plant. For example, the microorganism may be predicted to: improvenitrogen fixation; release phosphate from the soil organic matter;release phosphate from the inorganic forms of phosphate (e.g. rockphosphate); “fix carbon” in the root microsphere; live in therhizosphere of the plant thereby assisting the plant in absorbingnutrients from the surrounding soil and then providing these morereadily to the plant; increase the number of nodules on the plant rootsand thereby increase the number of symbiotic nitrogen fixing bacteria(e.g. Rhizobium species) per plant and the amount of nitrogen fixed bythe plant; elicit plant defensive responses such as ISR (inducedsystemic resistance) or SAR (systemic acquired resistance) which helpthe plant resist the invasion and spread of pathogenic microorganisms;compete with microorganisms deleterious to plant growth or health byantagonism, or competitive utilisation of resources such as nutrients orspace; change the colour of one or more part of the plant, or change thechemical profile of the plant, its smell, taste or one or more otherquality.

In one embodiment a microorganism or combination of microorganisms (thefirst set of one or more microorganisms) is selected from a pre-existingcollection of individual microbial species or strains that provides noknowledge of their likely or predicted benefit to a plant. For example,a collection of unidentified microorganisms isolated from plant tissueswithout any knowledge of their ability to improve plant growth orhealth, or a collection of microorganisms collected to explore theirpotential for producing compounds that could lead to the development ofpharmaceutical drugs.

In one embodiment, the microorganisms are isolated from the sourcematerial (for example, soil, rock, water, air, dust, plant or otherorganism) in which they naturally reside. The microorganisms may beprovided in any appropriate form, having regard to its intended use inthe methods of the disclosure. However, by way of example only, themicroorganisms may be provided as an aqueous suspension, gel,homogenate, granule, powder, slurry, live organism or dried material.The microorganisms may be isolated in substantially pure or mixedcultures. They may be concentrated, diluted or provided in the naturalconcentrations in which they are found in the source material. Forexample, microorganisms from saline sediments may be isolated for use inthis disclosure by suspending the sediment in fresh water and allowingthe sediment to fall to the bottom. The water containing the bulk of themicroorganisms may be removed by decantation after a suitable period ofsettling and either applied directly to the plant growth medium, orconcentrated by filtering or centrifugation, diluted to an appropriateconcentration and applied to the plant growth medium with the bulk ofthe salt removed. By way of further example, microorganisms frommineralized or toxic sources may be similarly treated to recover themicrobes for application to the plant growth material to minimise thepotential for damage to the plant.

In another embodiment, the microorganisms (including the first set ofone or more microorganism and/or the second set of one or moremicroorganisms) are used in a crude form, in which they are not isolatedfrom the source material in which they naturally reside. For example,the microorganisms are provided in combination with the source materialin which they reside; for example, as soil, or the roots, seed orfoliage of a plant. In this embodiment, the source material may includeone or more species of microorganisms.

In an embodiment, a mixed population of microorganisms is used in themethods of the disclosure.

In embodiments of the disclosure where the microorganisms are isolatedfrom a source material (for example, the material in which theynaturally reside), any one or a combination of a number of standardtechniques which will be readily known to skilled persons may be used.However, by way of example, these in general employ processes by which asolid or liquid culture of a single microorganism can be obtained in asubstantially pure form, usually by physical separation on the surfaceof a solid microbial growth medium or by volumetric dilutive isolationinto a liquid microbial growth medium. These processes may includeisolation from dry material, liquid suspension, slurries or homogenatesin which the material is spread in a thin layer over an appropriatesolid gel growth medium, or serial dilutions of the material made into asterile medium and inoculated into liquid or solid culture media.

In one embodiment, the material containing the microorganisms may bepre-treated prior to the isolation process in order to either multiplyall microorganisms in the material, or select portions of the microbialpopulation, either by enriching the material with microbial nutrients(for example, nitrates, sugars, or vegetable, microbial or animalextracts), or by applying a means of ensuring the selective survival ofonly a portion of the microbial diversity within the material (forexample, by pasteurising the sample at 60° C.-80° C. for 10-20 minutesto select for microorganisms resistant to heat exposure (for example,bacilli), or by exposing the sample to low concentrations of an organicsolvent or sterilant (for example, 25% ethanol for 10 minutes) toenhance the survival of actinomycetes and spore-forming orsolvent-resistant microorganisms). Microorganisms can then be isolatedfrom the enriched materials or materials treated for selective survival,as above.

In an embodiment of the disclosure endophytic or epiphyticmicroorganisms are isolated from plant material. Any number of standardtechniques known in the art may be used and the microorganisms may beisolated from any appropriate tissue in the plant, including for exampleroot, stem and leaves, and plant reproductive tissues. By way ofexample, conventional methods for isolation from plants typicallyinclude the sterile excision of the plant material of interest (e.g.root or stem lengths, leaves), surface sterilisation with an appropriatesolution (e.g. 2% sodium hypochlorite), after which the plant materialis placed on nutrient medium for microbial growth (see, for example,Strobel G and Daisy B (2003) Bioprospecting for microbial endophytes andtheir natural products. Microbiology and Molecular Biology Reviews 67(4): 491-502; Zinniel D K et al. (2002) Isolation and characterisationof endophytic colonising bacteria from agronomic crops and prairieplants. Applied and Environmental Microbiology 68 (5): 2198-2208). Inone preferred embodiment of the disclosure, the microorganisms areisolated from root tissue. Further methodology for isolatingmicroorganisms from plant material are detailed herein after.

It should be appreciated that the second set of microorganisms acquiredin step c) of a method of the disclosure may be isolated from a plant orplant material, surface or growth media associated with a selected plantusing any appropriate techniques known in the art, including but notlimited to those techniques described herein. However, in certainembodiments, as mentioned herein before, the microorganism(s) may beused in crude form and need not be isolated from a plant or a media. Forexample, plant material or growth media which includes themicroorganisms identified to be of benefit to a selected plant may beobtained and used as a crude source of microorganisms for the next roundof the method or as a crude source of microorganisms at the conclusionof the method. For example, whole plant material could be obtained andoptionally processed, such as mulched or crushed. Alternatively,individual tissues or parts of selected plants (such as leaves, stems,roots, and seeds) may be separated from the plant and optionallyprocessed, such as mulched or crushed. In certain embodiments, one ormore part of a plant which is associated with the second set of one ormore microorganisms may be removed from one or more selected plants and,where any successive repeat of the method is to be conducted, grafted onto one or more plant used in step a).

The methods of the disclosure may be described herein in terms of thesecond set of one or more microorganisms being isolated from theirsource material. However, unless the context requires otherwise, thisshould also be taken to include reference to the use of microorganismsin crude form in which they have not been isolated from the sourcematerial.

Plants

Any number of a variety different plants, including mosses and lichensand algae, may be used in the methods of the disclosure. In preferredembodiments, the plants have economic, social and/or environmentalvalue. For example, the plants may include those of use: as food crops;as fibre crops; as oil crops; in the forestry industry; in the pulp andpaper industry; as a feedstock for biofuel production; and/or, asornamental plants. In other embodiments, the plants may be economicallysocially and or environmentally undesirable, such as weeds. Thefollowing is a list of non-limiting examples of the types of plants themethods of the disclosure may be applied to:

Food Crops:

-   -   Cereals (maize, rice, wheat, barley, sorghum, millet, oats, rye,        triticale, buckwheat);    -   leafy vegetables (brassicaceous plants such as cabbages,        broccoli, bok choy, rocket; salad greens such as spinach, cress,        lettuce);    -   fruiting and flowering vegetables (e.g. avocado, sweet corn,        artichokes, curcubits e.g. squash, cucumbers, melons,        courgettes, pumpkins; solononaceous vegetables/fruits e.g.        tomatoes, eggplant, capsicums);    -   podded vegetables (groundnuts, peanuts, peas, soybeans, beans,        lentils, chickpea, okra);    -   bulbed and stem vegetables (asparagus, celery, Allium crops e.g        garlic, onions, leeks);    -   roots and tuberous vegetables (carrots, beet, bamboo shoots,        cassava, yams, ginger, Jerusalem artichoke, parsnips, radishes,        potatoes, sweet potatoes, taro, turnip, wasabi);    -   sugar crops including sugar beet (Beta vulgaris), sugar cane        (Saccharum officinarum);    -   crops grown for the production of non-alcoholic beverages and        stimulants (coffee, black, herbal and green teas, cocoa,        tobacco);    -   fruit crops such as true berry fruits (e.g. kiwifruit, grape,        currants, gooseberry, guava, feijoa, pomegranate), citrus fruits        (e.g. oranges, lemons, limes, grapefruit), epigynous fruits        (e.g. bananas, cranberries, blueberries), aggregate fruit        (blackberry, raspberry, boysenberry), multiple fruits (e.g.        pineapple, fig), stone fruit crops (e.g. apricot, peach, cherry,        plum), pip-fruit (e.g. apples, pears) and others such as        strawberries, sunflower seeds;    -   culinary and medicinal herbs e.g. rosemary, basil, bay laurel,        coriander, mint, dill, Hypericum, foxglove, alovera, rosehips);    -   crop plants producing spices e.g. black pepper, cumin cinnamon,        nutmeg, ginger, cloves, saffron, cardamom, mace, paprika,        masalas, star anise;    -   crops grown for the production of nuts e.g. almonds and walnuts,        Brazil nut, cashew nuts, coconuts, chestnut, macadamia nut,        pistachio nuts; peanuts, pecan nuts;    -   crops grown for production of beers, wines and other alcoholic        beverages e.g grapes, hops;    -   oilseed crops e.g. soybean, peanuts, cotton, olives, sunflower,        sesame, lupin species and brassicaeous crops (e.g.        canola/oilseed rape); and, edible fungi e.g. white mushrooms,        Shiitake and oyster mushrooms;

Plants Used in Pastoral Agriculture:

-   -   legumes: Trifolium species, Medicago species, and Lotus species;        White clover (T. repens); Red clover (T. pratense); Caucasian        clover (T. ambigum); subterranean clover (T. subterraneum);        Alfalfa/Lucerne (Medicago sativum); annual medics; barrel medic;        black medic; Sainfoin (Onobrychis viciifolia); Birdsfoot trefoil        (Lotus corniculatus); Greater Birdsfoot trefoil (Lotus        pedunculatus);    -   seed legumes/pulses including Peas (Pisum sativum), Common bean        (Phaseolus vulgaris), Broad beans (Vicia faba), Mung bean (Vigna        radiata), Cowpea (Vigna unguiculata), Chick pea (Cicer arietum),        Lupins (Lupinus species);    -   Cereals including Maize/corn (Zea mays), Sorghum (Sroghum spp.),        Millet (Panicum miliaceum, P. sumatrense), Rice (Oryza sativa        indica, Oryza sativa japonica), Wheat (Triticum sativa), Barley        (Hordeum vulgare), Rye (Secale cereale), Triticale (Triticum X        Secale), Oats (Avena fatua); Forage and Amenity grasses:        Temperate grasses such as Lolium species; Festuca species;        Agrostis spp., Perennial ryegrass (Lolium perenne); hybrid        ryegrass (Lolium hybridum); annual ryegrass (Lolium        multiflorum), tall fescue (Festuca arundinacea); meadow fescue        (Festuca pratensis); red fescue (Festuca rubra); Festuca ovina;        Festuloliums (Lolium X Festuca crosses); Cocksfoot (Dactylis        glomerata); Kentucky bluegrass Poa pratensis; Poa palustris; Poa        nemoralis; Poa trivialis; Poa compresa; Bromus species; Phalaris        (Phleum species); Arrhenatherum elatius; Agropyron species;        Avena strigosa; Setaria italic;    -   Tropical grasses such as: Phalaris species; Brachiaria species;        Eragrostis species; Panicum species; Bahai grass (Paspalum        notatum); Brachypodium species; and,    -   Grasses used for biofuel production such as Switchgrass (Panicum        virgatum) and Miscanthus species;

Fibre Crops:

-   -   cotton, hemp, jute, coconut, sisal, flax (Linum spp.), New        Zealand flax (Phormium spp.); plantation and natural forest        species harvested for paper and engineered wood fibre products        such as coniferous and broadleafed forest species;

Tree and Shrub Species Used in Plantation Forestry and Bio-Fuel Crops:

-   -   Pine (Pinus species); Fir (Pseudotsuga species); Spruce (Picea        species); Cypress (Cupressus species); Wattle (Acacia species);        Alder (Alnus species); Oak species (Quercus species); Redwood        (Sequoiadendron species); willow (Salix species); birch (Betula        species); Cedar (Cedurus species); Ash (Fraxinus species); Larch        (Larix species); Eucalyptus species; Bamboo (Bambuseae species)        and Poplars (Populus species).

Plants Grown for Conversion to Energy, Biofuels or Industrial Productsby Extractive, Biological, Physical or Biochemical Treatment:

-   -   Oil-producing plants such as oil palm, jatropha, soybean,        cotton, linseed;    -   Latex-producing plants such as the Para Rubber tree, Hevea        brasiliensis and the Panama Rubber Tree Castilla elastica;    -   plants used as direct or indirect feedstocks for the production        of biofuels i.e. after chemical, physical (e.g. thermal or        catalytic) or biochemical (e.g. enzymatic pre-treatment) or        biological (e.g. microbial fermentation) transformation during        the production of biofuels, industrial solvents or chemical        products e.g. ethanol or butanol, propane diols, or other fuel        or industrial material including sugar crops (e.g. beet, sugar        cane), starch-producing crops (e.g. C3 and C4 cereal crops and        tuberous crops), cellulosic crops such as forest trees (e.g.        Pines, Eucalypts) and Graminaceous and Poaceous plants such as        bamboo, switch grass, miscanthus;    -   crops used in energy, biofuel or industrial chemical production        via gasification and/or microbial or catalytic conversion of the        gas to biofuels or other industrial raw materials such as        solvents or plastics, with or without the production of biochar        (e.g. biomass crops such as coniferous, eucalypt, tropical or        broadleaf forest trees, graminaceous and poaceous crops such as        bamboo, switch grass, miscanthus, sugar cane, or hemp or        softwoods such as poplars, willows; and, biomass crops used in        the production of biochar;

Crops Producing Natural Products Useful for the Pharmaceutical,Agricultural NutraCeutical and Cosmeceutical Industries:

-   -   crops producing pharmaceutical precursors or compounds or        nutraceutical and cosmeceutical compounds and materials for        example, star anise (shikimic acid), Japanese knotweed        (resveratrol), kiwifruit (soluble fibre, proteolytic enzymes);

Floricultural, Ornamental and Amenity Plants Grown for their Aestheticor Environmental Properties:

-   -   Flowers such as roses, tulips, chrysanthemums;    -   Ornamental shrubs such as Buxus, Hebe, Rosa, Rhododendron,        Hedera    -   Amenity plants such as Platanus, Choisya, Escallonia, Euphorbia,        Carex    -   Mosses such as sphagnum moss

Plants Grown for Bioremediation:

-   -   Helianthus, Brassica, Salix, Populus, Eucalyptus

It should be appreciated that a plant may be provided in the form of aseed, seedling, cutting, propagule, or any other plant material ortissue capable of growing. In one embodiment the seed maysurface-sterilised with a material such as sodium hypochlorite ormercuric chloride to remove surface-contaminating microorganisms. In oneembodiment, the propagule is grown in axenic culture before being placedin the plant growth medium, for example as sterile plantlets in tissueculture.

Growth Medium

In one embodiment, the growth medium is a naturally occurring mediumsuch as soil, sand, mud, clay, humus, regolith, rock, or water. Inanother embodiment, the growth medium is artificial. Such an artificialgrowth medium may be constructed to mimic the conditions of a naturallyoccurring medium, however, this is not necessary. Artificial growthmedia can be made from one or more of any number and combination ofmaterials including sand, minerals, glass, rock, water, metals, salts,nutrients, water. In one embodiment, the growth medium is sterile. Inanother embodiment, the growth medium is not sterile.

The medium may be amended or enriched with additional compounds orcomponents, for example, a component which may assist in the interactionand/or selection of specific groups of microorganisms with the plant andeach other.

Further, in some embodiments, the medium may be amended or enriched withadditional compounds or components, for example, a compound orcomposition which provides any specific plant, microbial, or soilbenefit (e.g. molybdenum, humates) or modifies any aspect of any soilchemistry, microbial or animal properties e.g. the nitrificationinhibitor dicyaniamide (DCD), or microbial product(s) such asbiopesticide(s) or biofertilizer(s).

In certain embodiments of the disclosure, the growth medium may bepre-treated to assist in the survival and/or selection of certainmicroorganisms. For example, the medium may be pre-treated by incubatingin an enrichment media to encourage the multiplication of endogenousmicrobes that may be present therein. By way of further example, themedium may be pre-treated by incubating in a selective medium toencourage the multiplication of specific groups of microorganisms. Afurther example includes the growth medium being pre-treated to excludea specific element of the microbial assemblage therein; for examplepasteurization (to remove spore-forming bacteria and fungi) or treatmentwith organic solvents such as various alcohols to remove microorganismssensitive to these materials but allow the survival of actinomycetes andspore-forming bacteria, for example.

Methods for pre-treating or enriching may be informed by cultureindependent microbial community profiling techniques that provideinformation on the identity of microbes or groups of microbes present.These methods may include, but are not limited to, sequencing techniquesincluding high throughput sequencing and phylogenetic analysis, ormicroarray-based screening of nucleic acids coding for components ofrRNA operons or other taxonomically informative loci.

Growth Conditions

In accordance with the methods of the disclosure one or more plant issubjected to one or more microorganism and a growth medium. The plant ispreferably grown or allowed to multiply in the presence of the one ormore microorganism(s) and growth medium. The microorganism(s) may bepresent in the growth medium naturally without the addition of furthermicroorganisms, for example in a natural soil. The growth medium, plantand microorganisms may be combined or exposed to one another in anyappropriate order. In one embodiment, the plant, seed, seedling,cutting, propagule or the like is planted or sown into the growth mediumwhich has been previously inoculated with the one or moremicroorganisms. Alternatively, the one or more microorganisms may beapplied to the plant, seed, seedling, cutting, propagule or the likewhich is then planted or sown into the growth medium (which may or maynot contain further microorganisms). In another embodiment, the plant,seed, seedling, cutting, propagule or the like is first planted or sowninto the growth medium, allowed to grow, and at a later time the one ormore microorganisms are applied to the plant, seed, seedling, cutting,propagule or the like and/or the growth medium itself is inoculated withthe one or more microorganisms.

The microorganisms may be applied to the plant, seedling, cutting,propagule or the like and/or the growth medium using any appropriatetechniques known in the art. However, by way of example, in oneembodiment, the one or more microorganisms are applied to the plant,seedling, cutting, propagule or the like by spraying or dusting. Inanother embodiment, the microorganisms are applied directly to seeds(for example as a coating) prior to sowing. In a further embodiment, themicroorganisms or spores from microorganisms are formulated intogranules and are applied alongside seeds during sowing. In anotherembodiment, microorganisms may be inoculated into a plant by cutting theroots or stems and exposing the plant surface to the microorganisms byspraying, dipping or otherwise applying a liquid microbial suspension,or gel, or powder. In another embodiment the microorganism(s) may beinjected directly into foliar or root tissue, or otherwise inoculateddirectly into or onto a foliar or root cut, or else into an excisedembryo, or radicle or coleoptile. These inoculated plants may then befurther exposed to a growth media containing further microorganisms,however, this is not necessary. In certain embodiments, themicroorganisms are applied to the plant, seedling, cutting, propagule orthe like and/or growth medium in association with plant material (forexample, plant material with which the microorganisms are associated).

In other embodiments, particularly where the microorganisms areunculturable, the microorganisms may be transferred to a plant by anyone or a combination of grafting, insertion of explants, aspiration,electroporation, wounding, root pruning, induction of stomatal opening,or any physical, chemical or biological treatment that provides theopportunity for microbes to enter plant cells or the intercellularspace. Persons of skill in the art may readily appreciate a number ofalternative techniques that may be used.

It should be appreciated that such techniques are equally applicable toapplication of the first set of one or more microorganisms and thesecond set of microorganisms when used in step a) of a successive repeatof the method.

In one embodiment the microorganisms infiltrate parts of the plant suchas the roots, stems, leaves and/or reproductive plant parts (becomeendophytic), and/or grow upon the surface of roots, stems, leaves and/orreproductive plant parts (become epiphytic) and/or grow in the plantrhizosphere. In one preferred embodiment microorganism(s) form asymbiotic relationship with the plant.

The growth conditions used may be varied depending on the species ofplant, as will be appreciated by persons skilled in the art. However, byway of example, for clover, in a growth room one would typically growplants in a soil containing approximately ⅓^(rd) organic matter in theform of peat, ⅓rd compost, and ⅓rd screened pumice, supplemented byfertilisers typically containing nitrates, phosphates, potassium andmagnesium salts and micronutrients and at a pH of between 6 and 7. Theplants may be grown at a temperature between 22-24° C. in an 16:8 periodof daylight:darkness, and watered automatically.

For example, in the case of winter wheat varieties, mainly sown in theNorthern Hemisphere, it may be important to select plants that displayearly tillering after exposure of seed to a growth medium containingmicroorganisms under conditions of light and temperature similar tothose experienced by the winter wheat seed in the Northern Hemisphere,since early tillering is a trait related to winter survival, growth andeventual grain yield in the summer. Or, a tree species may be selectedfor improved growth and health at 4-6 months as these traits are relatedto the health and growth rate and size of trees of 10 years later, animpractical period product development using this disclosure.

Selection

Typically, following growth of the one or more plants in the presence ofone or more microorganisms, one or more plant is selected based on oneor more selection criterion. In one embodiment the plants are selectedon the basis of one or more phenotypic traits. Skilled persons willreadily appreciate that such traits include any observablecharacteristic of the plant, including for example growth rate, height,weight, colour, taste, smell, changes in the production of one or morecompounds by the plant (including for example, metabolites, proteins,drugs, carbohydrates, oils, and any other compounds). Selecting plantsbased on genotypic information is also envisaged (for example, includingthe pattern of plant gene expression in response to the microorganisms,genotype, presence of genetic markers). It should be appreciated that incertain embodiments, plants may be selected based on the absence,suppression or inhibition of a certain feature or trait (such as anundesirable feature or trait) as opposed to the presence of a certainfeature or trait (such as a desirable feature or trait).

Where the presence of one or more genetic marker is assessed, the one ormore marker may already be known and/or associated with a particularcharacteristic of a plant; for example, a marker or markers associatedwith an increased growth rate or metabolite profile. This informationcould be used in combination with assessment based on othercharacteristics in a method of the disclosure to select for acombination of different plant characteristics that may be desirable.Such techniques may be used to identify novel QTLs which link desirableplant traits with a specific microbial flora—for example matching plantgenotype to the microbiome type.

By way of example, plants may be selected based on growth rate, size(including but not limited to weight, height, leaf size, stem size,branching pattern, or the size of any part of the plant), generalhealth, and survival, as well as other characteristic, as describedherein before. Further non-limiting examples include selecting plantsbased on: speed of seed germination; quantity of biomass produced;increased root, and/or leaf/shoot growth that leads to an increasedyield (herbage or grain or fibre or oil) or biomass production; effectson plant growth that results in an increased seed yield for a crop,which may be particularly relevant in cereal crops such as wheat,barley, oats, rye, maize, rice, sorghum, oilseed crops such as soybean,canola, cotton, sunflower, and seed legumes such as peas, beans; effectson plant growth that result in an increased oil yield, which may beparticularly relevant in oil seed crops such as soybean, canola, cotton,jatropha and sunflower; effects on plant growth that result in anincreased fibre yield (e.g. in cotton, flax and linseed) or for effectsthat result in an increased tuber yield in crops such as potatoes andsugar beet; effects on plant growth that result in an increaseddigestibility of the biomass which may be particularly relevant inforage crops such as forage legumes (alfalfa, clovers, medics), foragegrasses (Lolium species; Festuca species; Paspalum species; Brachiariaspecies; Eragrostis species), forage crops grown for silage such asmaize and forage cereals (wheat, barley, oats); effects on plant growthwhich result in an increased fruit yield which may be particularlyrelevant to pip fruit trees (such as apples, pears, etc), berry fruits(such as strawberries, raspberries, cranberries), stone fruit (such asnectarines, apricots), and citrus fruit, grapes, figs, nut trees;effects on plant growth that lead to an increased resistance ortolerance disease including fungal, viral or bacterial diseases or topests such as insects, mites or nematodes in which damage is measured bydecreased foliar symptoms such as the incidence of bacterial or fungallesions, or area of damaged foliage or reduction in the numbers ofnematode cysts or galls on plant roots, or improvements in plant yieldin the presence of such plant pests and diseases; effects on plantgrowth that lead to increased metabolite yields, for example in plantsgrown for pharmaceutical, nutraceutical or cosmeceutical purposes whichmay be particularly relevant for plants such as star anise grown for theproduction of shikimic acid critical for the production ofanti-influenza drug oseltamivir, or the production of Japanese knotweedfor the extraction of resveratrol, or the production of soluble fibreand dietary enzyme products from kiwifruit, or for example increasedyields of “condensed tannins” or other metabolites useful for inhibitingthe production of greenhouse gases such as methane in grazing animals;effects on plant growth that lead to improved aesthetic appeal which maybe particularly important in plants grown for their form, colour ortaste, for example the colour intensity and form of ornamental flowers,the taste of fruit or vegetable, or the taste of wine from grapevinestreated with microorganisms; and, effects on plant growth that lead toimproved concentrations of toxic compounds taken up or detoxified byplants grown for the purposes of bioremediation.

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing),qRT-PCR (quantitative real time PCR).

In certain embodiments of the disclosure, selection for a combination ofplant traits may be desired. This can be achieved in a number of ways.In one embodiment, multiple rounds of iterative improvement for onetrait, e.g. superior growth, are maintained until an acceptable level ofgrowth is attained. Similar, but completely separate rounds of selectionare undertaken to identify microorganisms that can confer at leastdifferent desirable traits, for example for improved flower colour. Suchseparate rounds of selection may be performed using an iterative orstacking approach or a combination of separate methods could be used,with the microorganisms that result from those separate rounds ormethods being combined into a single composition. At this point themicroorganism(s) could be developed into a product containingcombinations of separately-fermented microorganisms each shown toimprove a different plant attribute. In a further embodiment, theseparately selected sets of microorganisms may be combined in sets oftwo or more and used in further methods of the disclosure. In anotherembodiment, the separately selected sets of microorganisms may beseparated into individual isolates and then individual isolates combinedin sets of two or more and used in further methods of the disclosure. Inone embodiment, the combined microorganisms are applied to the plantand/or growth medium in the same iterative cycle. For example, in onecombination, microorganisms able to improve plant growth are combinedwith microorganisms able to enhance flower colour. The combinedmicroorganisms are then added to a plant growth medium in which theplants are grown for a suitable period, under suitable conditions. Thedegree of growth and flower colour is assessed and microbes are isolatedfrom the best-performing plants for use in a succeeding iteration.Similar iterative rounds may be continued until an acceptable level ofplant growth and flower colour is attained. This approach will aid theselection of microbes that synergistically improve plant performance; byway on non-limiting example, improve plant growth and flower colour to adegree better than that achieved if the microorganisms are appliedsimply as a combination of two separately-selected sets.

Harvesting

Following selection, one or more plants are harvested and plant tissuesmay be examined to detect microorganisms forming associations with theplants (for example, endophytic, epiphytic or rhizosphericassociations).

The techniques described herein may be used in acquiring a second set ofmicroorganisms at the conclusion of a method of the disclosure or foruse in any successive repeat of the methods of the disclosure.

The one or more microorganisms may be isolated from any appropriatetissue of the plants selected; for example, whole plant, foliar tissue,stem tissue, root tissue, and/or seeds. In a embodiment, themicroorganisms are isolated from the root tissue, stem or foliar tissuesand/or seeds of the one or more plants selected.

In certain embodiments of the disclosure, the microorganisms may beacquired in crude form, in which they are not isolated from the sourcematerial in which they reside (such as plant tissue or growth media).

Where isolation of the microorganisms occurs, they may be isolated fromthe plants using any appropriate methods known in the art. However, byway of example, methods for isolating endophytic microbes may includethe sterile excision of the plant material of interest (e.g. root, stemlengths, seed), surface sterilisation with an appropriate solution (e.g.2% sodium hypochlorite), after which the plant material is placed onnutrient medium for microbial outgrowth, especially filamentous fungi.Alternatively, the surface-sterilised plant material can be crushed in asterile liquid (usually water) and the liquid suspension, includingsmall pieces of the crushed plant material spread over the surface of asuitable solid agar medium, or media, which may or may not be selective(e.g. contain only phytic acid as a source of phosphorus). This approachis especially useful for bacteria and yeasts which form isolatedcolonies and can be picked off individually to separate plates ofnutrient medium, and further purified to a single species by well-knownmethods. Alternatively, the plant root or foliage samples may not besurface sterilised but only washed gently thus includingsurface-dwelling epiphytic microorganisms in the isolation process, orthe epiphytic microbes can be isolated separately, by imprinting andlifting off pieces of plant roots, stem of leaves on to the surface ofan agar medium and then isolating individual colonies as above. Thisapproach is especially useful for bacteria and yeasts, for example.Alternatively, the roots may be processed without washing off smallquantities of soil attached to the roots, thus including microbes thatcolonise the plant rhizosphere. Otherwise, soil adhering to the rootscan be removed, diluted and spread out onto agar of suitable selectiveand non-selective media to isolate individual colonies of rhizosphericmicrobes. Further exemplary methodology can be found in: Strobel G andDaisy B (2003) Bioprospecting for microbial endophytes and their naturalproducts. Microbiology and Molecular Biology Reviews 67 (4): 491-502;Zinniel D K et al. (2002) Isolation and characterisation of endophyticcolonising bacteria from agronomic crops and prairie plants. Applied andEnvironmental Microbiology 68 (5): 2198-2208; Manual of EnvironmentalMicrobiology, Hurst et al., ASM Press, Washington D.C.

Methods for isolation may be informed by culture independent communityprofiling techniques that provide information on the identity andactivity of microbes present in a given sample. These methods mayinclude, but are not limited to, sequencing techniques including highthroughput sequencing and phylogenetic analysis, or microarray-basedscreening of nucleic acids coding for components of rRNA operons orother taxonomically informative loci.

In embodiments of the disclosure where two or more microorganism areisolated from plant material and then separated into individualisolates, any appropriate methodology for separating one or moremicroorganism from each other may be used. However, by way of example,microbial extracts prepared from plant material could be spread on agarplates, grown at an appropriate temperature for a suitable period oftime and the resulting microbial colonies subsequently selected andgrown in an appropriate media (for example, streaked onto fresh platesor grown in a liquid medium). The colonies may be selected based onmorphology or any other appropriate selection criteria as will beunderstood in the art. By way of further example, selective media couldbe used.

The one or more microorganisms may be harvested (including in isolatedor crude form) from the plants (including the rhizosphere as describedherein before) at any appropriate time point. In one embodiment they areharvested at any time after germination of the plant. For example, theycan be isolated from the period shortly after germination (wheresurvival in the first few days after germination is an issue, forexample with bacterial and fungal root and collar rots), then at anystage after that, depending on the timing required for a plant to growin order to evidence a discriminatory benefit that enables it'sselection from the plant population (for example, to discriminate saythe top 10 of 200 plants).

The inventor(s) has observed that different microorganisms may associatewith a plant at different stages of the plant's life. Accordingly,harvesting a plant at different time points may result in selection of adifferent population of microorganisms. Such microorganisms may be ofparticular benefit in improving plant condition, survival and growth atcritical times during its life.

In another embodiment of the disclosure, in the case of microorganismsthat form an association with a plant that allows vertical transmissionfrom one generation or propagule to the next (for exampleseed-endophytic or -epiphytic associations, or endophytic and epiphyticassociations with plants/propagules multiplied vegetatively) themicroorganisms may not be isolated from the plant(s). At the conclusionof a method of the disclosure, a target or selected plant itself may bemultiplied by seed or vegetatively (along with the associatedmicroorganisms) to confer the benefit(s) to “daughter” plants of thenext generation or multiplicative phase. Similarly, where a successiverepeat of the method is desired, plant material (whole plant, planttissue, part of the plant) comprising the set of one or moremicroorganisms can be used in step a) of any successive repeat.

Stacking

The inventor(s) envisage advantages being obtained by stacking the meansof selection (or the selection criteria) of plants in repeated rounds ofthe method of the disclosure. This may allow for acquiring a populationof microorganisms that may assist a plant in having a number ofdifferent desirable traits, for example.

In this embodiment of the disclosure the one or more microorganismsacquired from the one or more plants selected, as previously described,is used in a second round or cycle of the method. In the first round,one or more plants may have been selected based on biomass. In thesecond round, one or more plants may be selected based on production ofa particular compound. The microorganisms isolated from the second roundof the method may then be used in a subsequent round, and so on and soon. Any number of different selection criteria may be employed insuccessive rounds of the method, as desired or appropriate.

In one embodiment, the selection criteria applied in each repeat of themethod is different. However, in other embodiments of the disclosure,the selection criteria applied in each round may be the same. It couldalso be the same but applied at differing intensities with each round.For example, the selection criteria may be fibre levels and level offibre required for a plant to be selected may increase with successiverounds of the method. The selective criteria may increase or decrease insuccessive rounds in a pattern that may be linear, stepped orcurvilinear.

It should also be appreciated that in certain embodiments of thedisclosure, where one or more microorganism(s) forms an endophytic orepiphytic relationship with a plant that allows vertical transmissionfrom one generation or propagule to the next the microorganisms need notbe isolated from the plant(s). At the conclusion of a method of thedisclosure a target or selected plant itself may be multiplied by seedor vegetatively (along with the associated microorganisms) to confer thebenefit(s) to “daughter” plants of the next generation or multiplicativephase. Similarly, where a successive repeat of the method is desired,plant material (whole plant, plant tissue, part of the plant) comprisingthe set of one or more microorganisms can be used in step a) of thesuccessive repeat.

It should further be appreciated that two or more selection criterionmay be applied with each iteration of the method.

Microorganisms and Compositions Containing Same

In addition to the methods described herein before, the disclosurerelates to microorganisms selected, acquired, or isolated by suchmethods and compositions comprising such microorganisms. In its simplestform, a composition comprising one or more microorganisms includes aculture of living microorganism, or microorganisms in a live butinactive state(s), including frozen, lyophilised or dried cultures.However, the compositions may comprise other ingredients, as discussedbelow.

Thus, the disclosure provides, in some embodiments, for a compositioncomprising a microbial consortium that has been evolved to induce abeneficial property in a plant phenotypic trait.

The disclosure should also be understood to comprise methods for theproduction of a composition to support plant growth, quality and/orhealth or a composition to suppress or inhibit plant growth, qualityand/or health, the method comprising the steps of a method herein beforedescribed and the additional step of combining the one or moremicroorganisms with one or more additional ingredients.

Skilled persons will readily appreciate the types of additionalingredients that may be combined with the one or more microorganisms,having regard to the nature of the composition that is to be made, themicroorganisms to be used, and/or the method of delivery of thecomposition to a plant or its environment. However, by way of example,the ingredients may include liquid and/or solid carriers, microbialpreservatives, microbial activators that induce specific metabolicactivities, additives to prolong microbial life (such as gels andclays), wettable powders, granulated carriers, soil, sand, agents knownto be of benefit to microbial survival and the growth and general healthof a plant, peat, organic matter, organic and inorganic fillers, othermicroorganisms, wetting agents, organic and inorganic nutrients, andminerals.

Such compositions can be made using standard methodology having regardto the nature of the ingredients to be used. Compositions developed fromthe methods of the disclosure may be applied to a plant by any number ofmethods known to those skilled in the art. These include for example:sprays; dusts; granules; seed-coating; seed spraying or dusting uponapplication; germinating the seed in a bed containing suitableconcentrations of the composition prior to germination and planting outof the seedlings; prills or granules applied next to the seed or plantduring sowing or planting, or applied to an existing crop through aprocess such as direct drilling; application to plant cuttings or othervegetative propagules by dipping the cut surface or the propagule intoliquid or powdered microbial substrate prior to planting; application tothe soil as a “soil treatment” in the form of a spray, dust, granules orcomposted composition that may or may not be applied with plantfertilisers prior to or after sowing or planting of the crop;application to a hydroponic growth medium; inoculation into planttissues under axenic conditions via injection of compositions orotherwise inoculated via a cut in such tissues, for the subsequentestablishment of an endophytic relationship with the plant that extendsto the seed, or propagative tissues, such that the plant can bemultiplied via conventional agronomic practice, along with theendophytic microbe providing a benefit(s) to the plant.

In one embodiment, the disclosure provides a composition comprising oneor more of the microorganisms listed in table 4. In another embodiment,the disclosure provides a composition comprising one or moremicroorganisms listed in table 3. In another embodiment, the disclosureprovides a composition comprising one or more microorganisms listed intable 2.

In embodiments, the disclosure provides for a microbial consortium thatcontains at least one microbial species selected from the groupconsisting of: a member of the microorganisms of table 4, a member ofthe microorganisms of table 3, a member of the microorganisms of table2, and combinations thereof.

Disjunctive Associations

In one aspect, the disclosure provides for methods of assemblingdisjunctive microbial communities that are not associated with oneanother in a natural setting. That is, the present method is capable ofassembling microbial communities, or consortia, whose normal presence ina system would not bring the bacteria into close association with oneanother. In some aspects, the microbes are naturally located inphysically remote locations from one another. In other aspects, themicrobes may be located very close to one another physically, but theyinhabit different ecological niches that prevent their closeassociation. Thus, by utilizing the methods of the present disclosure,one is able to constructively bring together microbial species intoclose associations that are not naturally occurring.

In an embodiment, the methods of the present disclosure are able toidentify at least one microbial species that is not normally associatedwith a plant community in a natural setting; whereby, when the microbialspecies is brought into association with the plant the microbialspecies' abundance increases disproportionately greater than theabundance of other microbial species.

Consequently, the present methods are able to identify “invasive”microbial species that when brought out of their normal ecologicalhabitat and associated with a plant and substrate of interest, performbetter than microbial species that are “native” to the plant orsubstrate in question.

In a particular embodiment, the at least one microbial species that isidentified to perform better than expected, when associated with a plantof interest, is able to help the plant acquire a phenotypic trait ofinterest, e.g. biomass increase, sugar content, photosyntheticefficiency, water retention, growth on nutrient poor soils, etc.

Thus, by identifying microbes that are able to thrive outside of theirnatural ranges when associated with a particular plant and/or substrateof interest, the present disclosure can harness the ability of a microbeto be free from its coevolved constraints (e.g. from predators,landscape, nutrient constraints, etc.) and beneficially use that releaseon the microbe's population dynamics to increase a phenotypic trait ofinterest in the plant.

The present disclosure has discovered that microbes taken from theirnatural habitat and placed into association with a plant and/orsubstrate not normally encountered in their native range are able toincrease their abundance disproportionately more than the microbialassemblages presently associated with said plants and/or substrates. Themechanism of this microbial increase may be the ability of the microbesto outcompete the resident assemblages. The microbes may also benefitfrom association with the plant in a symbiotic way that the residentassemblages do not. Whatever the particular mechanism involved, thepresent disclosure provides a method to harness the tremendous amount ofmicrobial diversity present in the world's ecological regions and bringthat diversity to bear on increasing desirable plant phenotypic traits.

Disparate Geographic Locations

In particular embodiments, the microbial community assemblages derivedby the present methods are not naturally found in association with aparticular plant and/or substrate. In some aspects, the microbialspecies forming the microbial community are all from the same geographiclocation. In other aspects, each microbial species forming the microbialcommunity is from a different geographic location. A geographic locationcan be defined based upon the predominant soil type in a region, thepredominant climate in a region, the predominant plant community presentin a region, the distance between regions, the average rainfall in aregion, among others.

In a particular embodiment, at least one microbial species that is amember of the microbial community derived by the disclosed method isnative to, or was acquired from, a geographic region at least about: 1m, 10 m, 100 m, 1 km, 10 km, 100 km, 1000 km, 10,000 km, 20,000 km,30,000 km, or 40,000 km, from the location of the plant upon which aphenotypic trait is to be increased based upon the taught methods.

Genetically Modified Plants

In an aspect of the disclosure, methods are taught in which themicrobial communities produced herein are associated with increasing thephenotypic response of a genetically altered plant.

For example, various plants have been genetically associated withcommercial chemistries. Often, these genetic alterations enable theplant to be tolerant of the commercial chemical product, e.g. glyphosateresistance. Also, some plants have been genetically engineered toproduce toxins that repel pests, e.g. plants producing Bacillusthuringiensis toxins.

The present disclosure provides a method to create beneficial microbialconsortia that are correlated to improving a phenotypic trait of agenetically modified plant. The genetically modified plant may bemodified to tolerate a particular chemical product (or products in thecase of “stacked” chemical resistance) and/or may be geneticallymodified to produce a toxin. The present disclosure enablespractitioners to improve a phenotypic trait of interest in thesegenetically modified plants by deriving microbial consortia that aretailored to the genetically modified plants environment. For example,glyphosate resistant plant communities inhabit particular ecologicalconditions. Farmers growing these crops often employ no tillage systems,as the need to constantly till the soil for weed eradication isalleviated by the ability to directly spray glyphosate on the crops. Themicrobes that inhabit these types of systems will be confronted withmuch different ecological parameters than a microbe that was found in atraditional tillage agriculture system where the level of physicaldisturbance would be high and repetitive. The methods taught hereinenable the creation of microbial assemblages specifically adapted to theresident plant and its environment.

Methods of Producing Alternative Compositions

When microorganisms are cultured they may produce one or moremetabolites, which are passed into the media in which they reside. Suchmetabolites may confer beneficial properties to plants.

Accordingly, the disclosure also provides a method for selecting orproducing a composition capable of imparting one or more beneficialproperty to a plant, for example to support plant growth, quality and/orhealth, or for example to suppress or inhibit growth, quality and/orhealth of a plant, or to identify microorganisms that are capable ofproducing such a composition. In one embodiment, the composition issubstantially free of microorganisms.

In one embodiment, the method is for the selection of a compositioncapable of imparting one or more beneficial property to a plant andcomprises at least the steps of:

-   -   a) culturing one or more microorganism selected by a method as        herein before described in one or more media to provide one or        more culture;    -   b) separating the one or more microorganism from the one or more        media after a period of time to provide one or more composition        substantially free of microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition from step b);    -   d) selecting one or more composition of step c) if it is        observed to impart one or more beneficial property to the one or        more plants.

In another embodiment, the method is for the selection of a compositionwhich is capable of imparting one or more beneficial property to a plantand comprises the steps of:

-   -   a) culturing one or more microorganisms selected by a method of        the first aspect of the disclosure in one or more media to form        one or more culture;    -   b) inactivating the one or more culture of step a) to provide        one or more composition containing one or more inactivated        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition of step b);    -   d) selecting one or more composition from step c) if it is        observed to impart one or more beneficial property to the one or        more plants.

In one embodiment the method is for the selection of one or moremicroorganisms which are capable of producing a composition which iscapable of imparting one or more beneficial property to a plant andcomprises at least the steps of;

-   -   a) culturing one or more microorganism selected by a method of        the first aspect of the disclosure in one or more media to        provide one or more culture;    -   b) separating the one or more microorganism from the one or more        media in the one or more culture from step a) after a period of        time to provide one or more composition substantially free of        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition from step b);    -   d) selecting the one or more microorganisms associated with (or        in other words used to produce the) one or more composition        observed to impart one or more beneficial property to the one or        more plants.

Another method of the disclosure comprises at least the steps of:

-   -   a) culturing one or more microorganism in one or more media to        provide one or more culture;    -   b) separating the one or more microorganism from the one or more        media in the one or more culture after a period of time to        provide one or more composition substantially free of        microorganisms;    -   c) subjecting one or more plant (including for example seeds,        seedlings, cuttings, and/or propagules thereof) to the one or        more composition of step b);    -   d) selecting the one or more microorganisms associated with (or        in other words used to produce the) one or more composition        observed to impart one or more beneficial property to the one or        more plants; and,    -   e) using the one or more microorganisms selected in step d) in        step a) of a method of the first or eighth (and/or related)        aspects of the disclosure.

In one embodiment of the methods of the previous two paragraphs, step b)of the methods could be substituted with the step of b) inactivating theone or more culture of step a) to provide one or more compositioncontaining one or more inactivated microorganisms, and then using thiscomposition in step c) of the process.

The microorganisms can be inactivated, fixed, killed or destroyed usingany appropriate techniques know in the art. However, by way of example,one may use chemical agents and/or physical means to do so. In oneembodiment, the cells are lysed. In another embodiment, cells are fixedby chemical means, so as to render the organisms non-viable, butretaining their structural integrity.

In certain embodiments of these methods, the microorganisms are culturedin two or more (preferably a large number, for example, from at leastapproximately 10 to up to approximately 1000) mixed cultures using mediathat can support the growth of a wide variety of microorganisms. Anyappropriate media known in the art may be used. However, by way ofexample, growth media may include TSB (tryptic soy broth), Luria-Bertani(LB) broth, or R2A broth. In another embodiment, selective or enrichmentmedia which are able to support the growth of microorganisms with anarray of separate but desirable properties may be used. By way ofexample, the enrichment media referred to elsewhere herein may be used.

The microorganisms may be cultured in the media for any desired period.Following culture, the microorganisms are separated from the media andstored for later use. A separate composition also results. One or moreplants in a suitable growth medium are then subjected to the composition(using any known methodology, or methodology as described hereinbefore). After a period of time, growth of plants is assessed and plantsselected (as described herein before, for example). Plants arepreferably selected on the basis of size. However, other selectioncriteria as referred to herein may be used.

In one embodiment, the microorganism(s) producing the subset ofcompositions associated with the selected plants are recovered fromstorage. Two or more separate cultures of the microorganisms may then bemixed together and separated into two or more sub-cultures grown in twoor more different media. This process can be repeated iteratively asmany times as is deemed efficacious, with progressive steps refiningdown to fewer media and a narrower diversity of microorganisms until adesirable effect on the growth plants is achieved with a mixture ofmicrobes that can be identified, grown and stored indefinitely as astandard starting inoculum for the production the composition.

Thus, in some embodiments, a microbial consortium is produced containsmicrobes that work together for the common function of promoting orinducing a plant to express particular phenotypic trait of interest.

Compositions and consortium produced by the disclosure may be used orformulated on their own or combined with one or more additionalingredients.

It should be appreciated that the general methodology described may beapplicable to this aspect of the disclosure, including but not limitedto growth media, plants, microorganisms, timing, iterative processing,and combinations thereof.

Additional System Based Methodology

The following methodology may be applied to a method of the disclosurefor identifying one or more microorganisms as aforementioned.

FIG. 1 shows a system 10 according to an embodiment of the disclosure.System 10 includes requestors 11, request processor 12, growing facility13, database or library 14 and depository 15.

FIG. 2 provides a flow chart illustrating a method 20 according to anembodiment of the disclosure. The steps shown in FIG. 2 will bedescribed with reference to the system 10 shown in FIG. 1.

This system aspect of the disclosure is described in terms ofidentifying one or more microorganism that may impart one or moredesired properties to one or more plants, with particular reference tothe first, or eighth (and/or related) aspects of the disclosure.However, it should be appreciated that it is equally applicable to theidentification of one or more compositions that may impart one or moredesired property to one or more plant, or one or more microorganism thatproduces a composition that may impart one or more desired property toone or more plant, as herein before described, and summarised in theseventh (and/or related) and eighth (and/or related) aspects of thedisclosure. Accordingly, unless the context requires otherwise, whendescribing the embodiments of the disclosure in this section of thespecification, reference to the first aspect of the disclosure should betaken to also include reference to the seventh (and/or related) andeighth (and/or related) aspects of the disclosure, and reference to oneor more microorganism should be taken to include reference to one ormore composition.

The method begins at step 21 with a requestor 11 identifying a plant (ora class or group of plants). Reasons why particular plants or types ofplants may be identified will be apparent to those skilled in the art.However, by way of example, it may have been found that a plant noted ingeneral for having a high growth rate is growing at lower rates or notat all, there may simply be a desire to improve on existing growth ratesor there may be a desire to introduce a plant to a differentclimate/environment/geographical region. The disclosure is not limitedto conferring improvements to particular plant(s) and may be used toinhibit growth or otherwise adversely affect the plant(s).

At step 22, the requestor 11 sends the plant and/or the identity thereofto a request processor 12. The requestor 11 may provide further relevantinformation such as why or what properties they are seeking to improve.While only one request processor 12 is shown, it will be appreciatedthat more than one may be provided in the system 10.

Where a requestor 11 identifies a class or group of plants, more thanone plant variety may be evaluated. Alternatively or additionally,selection of a one or more plant variety may be made elsewhere withinthe system 10 based on the group or class identified, includingfollowing evaluation of different varieties including using differentmicroorganisms in accordance with methods of the disclosure.

Requests may conveniently be received over the internet via a webbrowser, although the disclosure is not limited thereto. Use of a webbrowser may additionally or alternatively be used to enable a requestor11 to view reports on the progress being made in response to theirrequest. For example, measures of growth may be provided.

At step 23, the request processor 12 receives and processes the request,essentially by initiating the performance of the method for theselection of one or more microorganism according to the first aspect ofthe disclosure. Note that the request processor 12 may or may notactively perform the method of the first aspect, or may only performparts thereof.

According to particular embodiments, the request processor 12 may act asan intermediary or agent between the requestor 11 and the parties ableto perform the method of the first aspect. Also, different arrangementsmay be made in response to different requests. For example, for onerequest, the environment around the request processor 12 may be suitablefor evaluating a particular plant but unsuitable for another, requiringthe assistance of a third party facility. This could be due to a desireto test in a particular soil type, altitude or climate. Other factorswill also be apparent although it is appreciated that “artificial”environments may be used. Furthermore, varying degrees of userinteraction may take place at the request processor 12. According to oneembodiment, a computer processor selects parameters or conditions for astudy based on data input by a requestor 11. As will be appreciated,providing a structured information request may help to effect this, andwhere necessary, reference may be made to databases including database14.

At step 24, parameters of the evaluation process are selected. Forexample, reference may be made to database 14 for microorganisms thatmay provide the desired improvement in the plant(s). While little datato date has been provided in the art on microorganisms having beneficialassociations with particular plant varieties, this will be improved uponthrough ongoing operation of the methods of the disclosure and stored indatabase 14. Other parameters such as plant type(s) and environmentalconditions may also be selected.

At step 25, the request (or portions thereof) and evaluation parametersare sent to growing facility 13 which may obtain suitable microorganismsfrom depository 15. These may or may not have been previouslyidentified. While only one growing facility 13 and one depository 15 areshown, it will be appreciated that the disclosure is not so limited.Furthermore, any two or more of request processor 12, growing facility13, database 14 and depository 15 may be co-located and/or under thesame control.

At step 26, a selection process is performed, preferably according tothe selection method of the first aspect.

At step 27, a response is sent to the request. A response may be sent tothe requestor 11 and/or to a third party and preferably includes atleast one of at least a subset of the results generated at step 26,identification of plant(s), plant(s), identification ofmicroorganism(s), microorganism(s), or plant(s) provided in associationwith microorganisms, namely those that have been shown to providebenefits at step 26.

At step 28, database 14 may be updated with results of the selectionprocess of step 26. This step may be performed prior to step 27,including periodically or at other various stages which the selectionprocess is conducted. Preferably, at least details of new beneficialassociations between plant(s) and microorganisms are recorded. It willbe appreciated that incompatible or less beneficial associations willalso preferably be recorded, thereby over time building a knowledgeframework of plants and microorganisms.

It will be appreciated that one or more of the steps of FIG. 2 may beomitted or repeated. For example, growing facility 13 may generateresults at step 26 and in response thereto, one or more of steps 21 to26 may be repeated.

Thus, the disclosure provides means and methods to improve plant(s) (orgrowth or other characteristics thereof). This is achieved by enabling arequestor 11 in a first geographical region (e.g. country) or otherwisedefined environment (e.g. by parameters or characteristics affectinggrowing conditions such as such soil salinity or acidity) to accessmicrobiological biodiversity not present or of limited presence in thefirst region for the purposes of plant improvement in the first oranother region. The other region may be or in a foreign country but maybe otherwise defined by characteristics of that environment that affecta plant rather than being defined by political boundaries. Consequently,the disclosure may enable a requestor to obtain the beneficial effectsof a particular microorganism(s) on a particular plant(s) in a firstregion, even though such microorganism(s) may not be present or are oflimited presence in the first region.

An example implementation of the system embodiment of the disclosure isprovided below.

-   -   1. A company in say New Zealand (home company), enters into a        contractual relationship with a second, say overseas, company        (overseas company).    -   2. The overseas company agrees to send seeds, cuttings or other        plant propagules (foreign cultivar) to the home company from        plant cultivars adapted to the environment(s) in its own, or        other foreign countries, in order to gain access to elements of        New Zealand's terrestrial and marine microbial biodiversity that        are able to form beneficial plant-microorganism associations        with the foreign cultivar.    -   3. The nature of the benefit may encompass increased plant        productivity, for example through any one or more of but not        limited to: increased root or foliar mass, or through an        increase in efficiency in nutrient utilisation through nitrogen        fixation by diazatrophs such as Klebsiella or Rhizobium, or        through release of plant nutrients from the soil, such as        phosphates released soil through the production of microbial        phytases, or through improvements in plant phenotype for example        date of flowering, or changes in physical form e.g. colour,        frequency of root or foliar branching, or changes in chemical        profile including compounds associated with taste, smell or        properties which make the plant suitable for a particular        purpose.    -   4. In New Zealand, the home company identifies which indigenous        microorganisms can form an association with the foreign plant by        exposing the seed to the microorganisms, with or without        knowledge of their likely effects on the plant, by the method of        germinating the seed and growing the plant in a growing material        that ensures contact of the plant during its growth with        indigenous microorganisms via seed coating, direct inoculation        into the seed or germinating seedling and/or contamination of        the growing medium. The disclosure is not limited to this        arrangement or methodology. For example, it may be apparent that        microorganisms present in soil other than in New Zealand may        provide benefits and testing may be conducted in such regions in        addition to or instead of New Zealand. Also, artificial        environments may be created. Referring to the immediately prior        example, this may be achieved by obtaining soil and/or        microorganisms from such regions and conducting the tests in say        New Zealand. As will be apparent, such embodiments may include        provision for artificial control of climatic conditions among        other parameters. Thus, the disclosure is not limited to        conducting testing in a region based on its indigenous        microorganisms—the microorganisms may be artificially introduced        so as to conduct the testing elsewhere than in the        microorganisms' natural environment.    -   5. The period of growth and the physical conditions under which        they take place may vary widely according to plant species and        specific plant improvement traits, including based on parameters        desired or specified by the overseas company. After the relevant        period of plant growth the nature of possible        plant-microorganisms associations may be determined by        microbiological assessment to determine whether microorganisms        have formed an endophytic, epiphytic or rhizospheric association        with the foreign crop.    -   6. Where such association(s) are demonstrated the microorganisms        form a collection of (say New Zealand indigenous) microorganisms        able to associate with the (say foreign) crop or plant.    -   7. In one embodiment of the disclosure, microbial isolates of        the collection may, for example, be coated on to seeds,        inoculated into seeds or seedlings, or inoculated into a growing        medium that may or may not be sterile.    -   8. After a suitable period the plants are assessed for improved        root and foliar growth or other desired characteristics designed        to identify the plant-microorganism associations most able to        provide benefit to the plant in the manner desired by the        overseas company.    -   9. Examples of selection criteria are provided herein before,        and where identical parameters of the second, overseas        environment are not present in the home or test region (i.e.,        New Zealand in the example), similar parameters most similar to        those in the overseas environment and that may be considered        acceptable to the overseas company may be selected. As mentioned        in 4 above, the disclosure also includes introducing foreign        material or creating otherwise artificial conditions in the home        or test region.    -   10. The steps involving growing one or more plant in the        presence of one or more microorganism, selecting one or more        plants with desired characteristics, and acquiring the        microorganism(s) forming an association with the plant will be        repeated one or more time.    -   11. Elite microorganisms providing commercially-significant        benefit to the growth of the foreign cultivar are identified by        this process and may be shipped to the overseas company for        further testing and selection in the foreign environment.    -   12. In a further embodiment the overseas company will agree that        microorganisms found on, or in, the seed, cuttings or propagules        of the foreign cultivar will be added to the collection of the        home company to enlarge the collection for use both on that        cultivar or on other foreign cultivars received for similar        testing from other companies.

In an alternative embodiment, the microbial isolates able to formplant-microorganism associations with the foreign cultivar i.e., thecollection, are sent to the second company for testing and selection,such that items 7-11 above are performed by and/or in the grounds of thesecond company. This may be performed by or under the control of thefirst company.

As a further embodiment, rather than identifying and using predeterminedmicroorganism(s) of a collection, the home company may simply expose theseed to indigenous microorganisms, with or without knowledge of theirlikely effects on the plant, for example by germinating the seed andgrowing the plant in a growing material that ensures contact of theplant during its growth with indigenous microorganisms via seed coating,direct inoculation into the seed or germinating seedling and/orcontamination of the growing medium or otherwise. As will be apparent,the home company may additionally or alternatively arrange for similartesting in other regions, where the same or different microorganisms maybe present. The period of growth and the physical conditions under whichthey take place may vary widely according to plant species and specificplant traits desired by the overseas company. After a period of plantgrowth the nature of possible plant-microorganism associations may bedetermined in a similar manner to that described above.

EXAMPLES

The disclosure is now further described by the following non-limitingexamples.

Example 1: Identification of Microorganisms Able to Improve the SugarContent of Forage Crops Such as Ryegrass

Step 1. Untreated ryegrass seeds are planted in a wide variety of soilsin small pots. Soils may include additional amendments comprising purecultures of microorganisms, mixtures of microorganisms or materialscontaining microorganisms derived from other sources.

Step 2. After a suitable period of growth, say 1 month, the plants arewashed out of the soil, and the microorganisms isolated from roots andstems/foliage, either as individual isolates in pure culture, or asmixed populations e.g. as a microbial suspension from an aqueous rootcrush and/or a stem/foliar crush.

Step 3. The microorganisms are then added to a plant growth medium intowhich untreated ryegrass seeds are planted. Alternatively, themicroorganism(s) are mixed into a suitable seed coating material e.g. agel, and coated onto seeds before being planted into a similar plantmedium. Alternatively, the seeds are geminated and then exposed to themicroorganisms for a short period (usually between 1-24 hours tomaximise the chance that the microbes may form an endophytic orepiphytic association with the germinating plant) and then planted intoa similar growth medium. In each of these cases the growing medium maybe initially sterile, although this is not essential.

Step 4. After a period of suitable growth, e.g. 4-6 weeks, foliar growthis assessed and sugar content of crushed foliage determined using arefractometer or other method known to a person skilled in the art. Theplants with the highest values for both foliar yield and/or sugarcontent are selected, and their root and foliar microorganisms isolatedand prepared as in Step 2. The process from step 2 to step 3 may then berepeated iteratively, with or without modification of the selectioncriteria for sugar content relative to foliar yield.

Step 5. After this iterative process has been conducted to the point atwhich improvement in the sugar content is deemed to be sufficient, thebest-performing plants are selected and the microorganisms associatedwith them are isolated and used to develop a commercial product thatimproves sugar content of ryegrass.

In an aspect, the product produced from the selected microorganisms is amicrobial consortium that is specialized to improve the sugar content ofryegrass. In another aspect, a composition comprising a microbialconsortium specialized to improve the sugar content of ryegrass isproduced.

Example 2: Identification of Microorganisms Able to Improve theTillering of Grain Crops Such as Wheat

In the case of winter wheat varieties, mainly sown in the NorthernHemisphere, it may be important to select plants that display earlytillering after exposure of seed to a growth medium containingmicroorganisms under conditions of light and temperature similar tothose experienced by winter wheat seed in the Northern Hemisphere, sinceearly tillering is a trait related to winter survival, growth, andeventual grain yield in the summer.

Step 1. Untreated wheat seeds are planted in a wide variety of soils ormicrobial substrates in small pots. Soils may include additionalamendments comprising pure cultures of microorganisms, mixtures ofmicroorganisms or materials containing microorganisms that are derivedfrom other sources.

Step 2. After a suitable period of growth period, say 1 month, theplants are washed out of the soil, and the microorganisms isolated fromroots and stems/foliage, either as individual isolates in pure culture,or as mixed populations e.g. as a microbial suspension from an aqueousroot crush and/or a stem/foliar crush.

Step 3. The microorganisms are then added to a plant growth medium intowhich untreated wheat seeds are planted. Alternatively, themicroorganism(s) are mixed into a suitable seed coating material e.g. agel, and coated onto seeds before being planted into a similar plantmedium. Alternatively, the seeds are geminated and then exposed to themicroorganisms for a short period (usually between 1-24 hours tomaximise the chance that the microbes may form an endophytic orepiphytic association with the germinating plant) and then planted intoa similar growth medium. In each of these cases, the growing medium maybe initially sterile, although this is not essential.

Step 4. Tillering is assessed after a suitable period of growth. Plantswith the first tillers and/or the greatest number of tillers over aspecific time period are selected, and their root and foliarmicroorganisms isolated and prepared as in Step 2. The process from step2 to step 3 may then be repeated iteratively, with or withoutmodification of the selection criteria for tillering relative toeventual grain yield.

Step 5. After this iterative process has been conducted to the point atwhich improvement in tillering is deemed to be sufficient, thebest-performing plants are selected and the microorganisms associatedwith them are isolated and used to develop a commercial product thatimproves the speed and degree of wheat tillering.

In an aspect, the product produced from the selected microorganisms is amicrobial consortium that is specialized to improve the speed and degreeof wheat tillering. In another aspect, a composition comprising amicrobial consortium specialized to improve the speed and degree ofwheat tillering is produced.

Example 3: Use of an Accelerated Microbial Selection Process to SelectSeed-Borne Endophytes Conveying a Beneficial Crop Trait

Forage grasses expressing beneficial traits such as insect-resistanceand improved tolerance to both biotic and abiotic stressors via strainsof the seed-borne fungus Neotyphodium sp. have been widely adopted byfarmers in New Zealand and elsewhere.

It would be desirable to extend the benefits of traits similar to thoseexpressed by this seed-borne fungus and other similar species in thefungal family, to a broader range seed-borne endophytic microbes therebyproviding access to a much wider range of beneficial crop traits.

Step 1. Untreated ryegrass seeds are planted in a wide variety of soilsin small pots. Soils may include additional amendments comprising purecultures of microorganisms, mixtures of microorganisms or materialscontaining microorganisms that derived from other sources.

Step 2. After a suitable period of growth, the plants are washed out ofthe soil, surface sterilised with a combination of ethanol and sodiumhypochlorite or other methods known to people skilled in the art, andthe endophytic microorganisms (endophytes) isolated from internaltissues of roots and stems/foliage and seeds, either as individualisolates in pure culture, or as mixed populations e.g. as a microbialsuspension from an aqueous root crush and/or a stem/foliar crush.

Step 3. The endophytic microorganisms are then added to a plant growthmedium into which pre-germinated surface-sterilised ryegrass seeds areplanted (seeds checked for sterility by germinating on nutrient agarplates). Alternatively, the microorganism(s) are mixed into a suitableseed coating material e.g. a gel, and coated onto surface-sterilisedseeds before being planted into a similar plant medium. Alternatively,the surface-sterilised seeds are geminated on nutrient agar plates,checked for sterility and then exposed to the microorganisms for a shortperiod (usually between 1-24 hours to maximise the chance that themicrobes may form an endophytic or epiphytic association with thegerminating plant) and then planted into a similar growth medium. Ineach of these cases the growing medium may be initially sterile,although this is not essential and further microorganisms may be appliedto the growth medium and/or plant.

Step 4). After a period of suitable growth, e.g. 4-6 weeks, plants areassessed for expression of the desired phenotype. Phenotypes may includeimproved color, plant form, metabolite expression, or the like.

Step 5). Selected plants are permitted to grow onward to the point ofseed set. At this stage a subset of seeds from each plant may bescreened for endophyte carriage using culture dependent or independentmethods. The remaining seeds from plants yielding positive results inthe screen are germinated and planted without microbial addition in afurther round of selection to enrich for endophyte carriage and theability to transmit the desired phenotype as described in steps 3-5.

Alternatively, endophytic microbes may be acquired from a subset ofseeds from each plant either as isolates from surface sterilised seedsor as explants, or as a microbial suspension prepared, for example, bycrushing the surface sterilised seed in aqueous solution. Isolates andpreparations are used as an inoculum for plants arising from surfacesterilised seeds as described in step 3.

In a further variation of the method, the selection for seedtransmission of the trait may take place in the following generation bysurface sterilising a subset of seeds (with or without prior screening)from the selected plants of the prior generation and allowing them togerminate and grow on for the period at which point phenotypic screeningis conducted as generally described in steps 3 and 4 (i.e. prior to seedset). Plants exhibiting the desired phenotype in this generation (i.e.by seed transmission), are selected and either tissue explants areprepared, and/or microbes isolated from plant tissues, and/or crudemicrobial suspensions made by crushing the surface foliage or roots inan aqueous solution. One or a combination of these preparations are usedas an inoculum for further iterative rounds of growth and selection andseed harvest, as described in steps 3-5. Alternatively, the remainingseeds of plants exhibiting the desired seed-borne trait may begerminated and planted without microbial addition in a further round ofselection to enrich for endophyte carriage and the ability to transmitthe desired phenotype as described in steps 3-5.

Step 6). At the end of successive rounds of this iterative process, asdetermined by the generation of a desired seed-borne phenotype, the bestseed lines are selected for commercial assessment and cultivardevelopment.

Example 4: Use of an Accelerated Microbial Selection Process to AcquireMicrobes Capable of Improving the Growth of Ryegrass (Loliism Perenne)

Ryegrass is often grown in fertile soil and is an important crop inforage production. It would be desirable therefore to use the process ofdirected selection to identify a group of microbes that are able toincrease the biomass of ryegrass in a fertile substrate withoutexperimentally-imposed selection pressures.

Seventy-three soil samples (treatments) from the North Island of NewZealand were used as a source of microbial diversity for the start ofthe process. Soil samples were mixed with sand:vermiculite (1:1 or 1:2)as required to increase drainage and volume. Samples were placed in tenreplicate 28 ml tubes and planted with ryegrass seeds (Loliism perennecultivar One50, nil endophyte). Seeds were watered with a misting hoseuntil germinated, then showered to saturation three times weekly withadditional watering as required to prevent seedlings drying out. Forstandard growing conditions see Table 1.

TABLE 1 Standard growing conditions Variable Conditions Watering Threetimes each week to saturation with water or synthetic fertilizerTemperature Constant 22-24° C. Daylight period 16 hr followed by 8 hrdarkness Seed sterilization 15 min in 1-2% sodium hypochlorite followedby 30 min quenching in sodium thiosulphate as described by Miche andBalandreau (2001) Volume of soil/replicate 28 ml Randomisation Alltreatment replicates and controls were spatially randomised

Round 1 Selection

Sixty days after sowing (DAS) four plants from each sample were selectedand processed to provide the microbial inoculum for the first round ofselection. Foliage was cut 2 cm above the substrate and discarded. Theroots and attached stems were shaken free of soil, washed to remove mostsoil fragments and drained before the roots and stems were combined inplastic bags. This material was then crushed within the bag with 10 mlsof water added to suspend the root material. The liquid portion of theresulting suspension was used as the initial microbial inoculum.Surface-sterilised seeds were soaked for one hour in 1 ml of the rootsuspension for each sample. Soaked seeds were then planted into 28 mltubes (15 reps for each treatment) containing potting mix (Kings PlantBarn, New Zealand; granulated bark, peat moss, pumice, and slow-releasefertilisers) moistened with tap water. The remaining root suspension wasmade up to a sufficient final volume with SDW and 2 ml was pipetted overthe planted seeds. After planting, the seeds were thinly covered withfresh dry substrate. Pots were subsequently watered with tap water 3times weekly.

Round 2 Selection

At 118 DAS the foliage was harvested, weighed and treatments selected toprovide microbial inoculum for the second round of selection. Only the 8largest plants from each of the 21 treatments with the greatest meanfoliar weight of the original 73 treatments were chosen for processing.In addition four composite treatments of four plants each were createdfrom the sixteen individual plants with the greatest foliar biomass.Foliage was cut 2 cm above substrate level and weighed. The roots andbasal stems of each plant were shaken free of substrate then rinsed,combined in plastic bags, crushed and used to inoculate the second roundof selection in the same way as described for selection round 1, withthe exception that 30 replicates were planted for each treatment and thefinal volume of inoculum was 65 mls.

Round 3 Selection

Plants from the second round of selection were harvested at 39 DAS.Foliage was cut 2 cm above substrate, weighed and discarded. The threelargest plants from the top 15 treatments were selected to create theinoculum for selection round 3. Roots and stems were crushed asdescribed above and used for 30 replicates of each treatment.

Microbial Isolation

Foliage from round 3 selection was harvested and weighed at 63 DAS andthe largest plants from the five treatments with the greatest meanfoliar weights were selected to provide inoculum for microbialisolations. The roots and 2 cm stems were rinsed and then crushed inplastic bags as described previously. A small volume of the inoculum wasdrawn off to make a ten-fold dilution series plated on R2A. Pieces ofcrushed root from each of the preparations were also inoculated into 10ml N-deficient semi-solid malate (NDSM) medium (Eckford et al, 2002.After 2-4 days incubation at room temperature the resulting pellicleswere drawn off and spread onto R2A agar for isolation of individualcolonies. A selective isolation step for actinomycetes was performed inwhich ethanol was added to the root suspension at a final concentrationof 25%, incubated at room temperature (RT) for 30 min then plated onR2A. For fungal isolations, pieces of crushed root were embedded inmolten PDA (cooled to 45° C.). After 24-72 hr incubation at 25° C. R2Aand PDA plates were examined under a dissecting microscope. Bacterialand fungal colonies were assessed for abundance, grouped according tomorphology and representative isolates were picked and streaked ontofresh R2A or PDA plates. Standard methods were used to identify isolatesto species level by DNA extraction, PCR amplification and sequencing of16S rRNA genes (bacteria) or ITSS region (fungi).

Microbial Evaluation

Microbial evaluation was performed on 61 individual isolates and 28consortia chosen on the basis of abundance, diversity, and speciescharacteristics. Selected isolates were spread on R2A (bacteria) or PDA(fungi), incubated at 25° C. for 72 hours then scraped off the agarsurface with added SDW into sterile containers. Bacteria were harvestedinto 2 ml SDW. Fungi were sieved through a sterile tea strainer with5-10 ml SDW to remove clumps of mycelia and pieces of attached agar.Serial dilutions of the harvested cells were plated and incubated at 25°C. for 24 hours to estimate the number of colony forming units (CFU) ineach suspension. Dilution volumes corresponding to 1×10⁷ (bacteria) and1×10³ (fungi) CFU per ml were calculated from these plate counts.Ryegrass seeds (One50 nil endophyte) were soaked for one hour in microbesuspensions then individually planted in 28 ml tubes containingmoistened potting mix. Two millilitres of isolate suspension waspipetted over the seeds which were then covered with substrate. Allplants were subsequently watered with tap water 3 times weekly. Foliagewas cut and weighed at 41 DAS. Roots were washed, blotted dry andweighed. The microbial treatments that resulted in plant biomass gainsof at least 5% over the microbe-free controls are shown in Table 2.

TABLE 2 Microbial treatments associated with increased ryegrass biomassTreatment % IOC % IOC % IOC BDNZ# FW RW BM ID 58918 22.9 25.4 23.5Microbacterium ginsengiterrae 58900 25.3 7.7 21.3 Bacillus cereus 5891321.5 16.6 20.4 Microbacterium oxydans Consortium 18.9 5.1 15.8 Rhizobiumpusense, Curtobacterium ginsengisoli 59084 21.8 −7.4 15.2 Penicilliumdaleae 58894 13.3 4.3 11.3 Brevundimonas vesicularis 58910 13.6 2.3 11.1Aeromicrobium ponti 58895 11.2 5.5 9.9 Microbacterium hydrocarbonoxydans58911 8.9 5.3 8.1 Sphingopyxis chilensis 58950 7.8 8.4 7.9 Arthrobacterkeyser 59088 13.6 −14.6 7.3 Penicillium melinii 58892 8.4 0.4 6.6Rhizobium grahamii 58948 8.6 −2.2 6.2 Brevundimonas vesicularisConsortium 5.1 9.7 6.2 Rhizobium pusense, Curtobacterium ginsengisoli,Herbaspirillum rubrisubalbicans 58891 6.7 −0.2 5.1 Rhizobium etliConsortium 4.9 5.6 5.0 Exiguobacterium indicum, Mesorhizobium amorphae,Brevundimonas vesicularis, Arthrobacter keyser FW = fresh foliar weight;RW = fresh root weight; BM = plant biomass (roots + foliage) Italicsindicate a significant IOC (increase over controls; Fisher’s LSD)ID-Putative identification based on closest sequence match in RDPIIand/or NCBI databases

The three microbial treatments that resulted in a significant increasein foliar weights (Fisher's LSD) were all isolated from the site thatproduced the greatest increase in foliar weight in the third selectionround.

These results provide evidence that the method for directed selection ofmicrobes, also referred to herein as accelerated microbial selection,described by the present disclosure is capable of identifying a set ofmicrobes that significantly improve the growth of ryegrass grown underfavorable conditions.

Furthermore, as indicated by Table 2, the methods are able to identifymicrobial consortia that significantly improve the growth of ryegrassgrown under non-selective conditions.

Example 5: Use of an Accelerated Microbial Selection Process to IdentifyMicrobes Able to Improve the Water-Soluble Carbohydrate Content of Basil(Ocium Basilicum)

Soil samples from 43 sites in the North Island of New Zealand were usedas a source of microbial diversity for this process.

Samples were mixed with sand:vermiculite (1:2) as required to increasedrainage and volume. Each sample was used to fill five replicate 28 mltubes which were planted with 3-5 basil seeds (Ocium basilicum, varietySweet Genovese) per tube. Seedlings were germinated in a plant growthroom under conditions described in Table 1. Watering was carried outwith tap water as required to prevent wilting.

Approximately 14 DAS the plants were harvested and the foliage cut anddiscarded. For each sample the basal stems and roots were shaken free ofsoil, rinsed in sterile distilled water (SDW) and the replicatescombined in a plastic bag. The plant material was then crushedthoroughly within the plastic bags. 10 ml SDW was added to the crushedroots and the resulting suspension used as the microbial inoculum forthe first selection round.

Basil seeds were soaked for a minimum of one hour in the root extractthen planted into 28 ml tubes containing potting mix (40% v/v peat, 30%composted pine bark, 30% fine pumice, adjusted to pH 6.1 with lime)moistened with 6 ml of liquid fertiliser (Miracle-Gro, Scotts AustraliaPty Ltd). The remaining root suspension was diluted with 40 ml of SDWand 2 ml was pipetted over the seeds. Ten replicate tubes were preparedfor each sample alongside a set of 20 no-microbe controls that wereprepared using seeds soaked in sterile distilled water. All tubes wererandomised across racks. Seedlings were germinated in a plant growthroom under conditions described above. After germination each tube wasweeded to leave one randomly selected seedling.

Round 1 Selection

At 20 DAS half of the plants from each treatment were randomly selectedfor harvest. The remainder of the plants were retained in the growthroom for preparation of extracts to inoculate the second round ofselection. Plants selected for harvest were removed from the pots,washed to remove adherent potting mix, dried on paper towels and weighedbefore being placed into a 2 ml tube containing a single stainless steelball bearing. Samples were then frozen at −20□° C. pending analysis forwater soluble carbohydrate.

The concentration of water soluble carbohydrate (WSC) in plant extractswas determined using the anthrone method as generally described by Yemmand Willis (Biochem. J. 1954, 57: 508-514). Whole-plant extracts wereprepared by bead beating for 2 minutes at 22 hz. One mL of steriledistilled water was then added to each sample. After mixing, 0.5 mL ofthe liquid suspension was transferred a 96-well microtube block whichwas placed in a boiling water bath for 30 minutes. Each block was thentransferred to a cold water bath for five minutes followed bycentrifugation at 3000 rcf for 10 minutes to pellet debris. Supernatantswere recovered, diluted 1:25 in SDW, and 40 μL, samples transferred tonew 96-well microtube blocks. Samples were then overlaid with 200 μL, offreshly-prepared anthrone solution (2 mg/mL in 70% sulphuric acid).Blocks were cooled for 5 minutes in an ice-cold water bath, mixed byinversion, placed in a boiling water bath for 60 seconds, thenimmediately returned to the cold water bath. Once cooled, a 100 ulsample of each reaction was transferred to a flat-bottomed microtitretray and the absorption measured at 600 nm on a SpectraMax M5espectrophotometer. Glucose standards were prepared in ultra-pure waterand processed as per plant extracts to generate a calibration curve.Results are reported in glucose equivalents (mg) per gram of planttissue.

Twenty of the 43 treatments yielded a positive increase in median sugarcontent over the no-microbe control.

Round 2 Selection

The 13 treatments yielding the greatest median sugar content wereselected for the second selection round. Microbial extracts wereprepared from the remaining 5 plants in each treatment and applied tobasil seeds according to the procedure described above with theexception that the number of replicates was increased to 30 for eachtreatment and 60 for no-microbe controls.

Fifteen days after sowing (DAS) 15 of the plants from each treatmentwere harvested. The remainder of the plants were retained in the growthroom for subsequent isolation experiments. Plants selected for harvestwere removed from pots and processed for analysis of water solublecarbohydrate as described previously, with the exception that theanthrone solution was prepared in 80% sulphuric acid to reduce formationof precipitates.

Eight of the 13 treatments yielded a positive increase in median sugarcontent over the no-microbe control. At this point the rounds ofiterative selection were concluded and microbial isolations wereperformed.

Microbial Isolation

Bacteria and fungi were isolated from up to five of the remaining plantsfrom each of the seven treatments with the greatest median WSC. For eachtreatment, the roots and lower 1 cm of stem material from each plantwere shaken free of substrate and rinsed in sterile distilled water thendivided into two portions. One portion was surface sterilized in 6.6%Dettol® (active ingredient: chloroxylenol 4.8%) for 1 minute followed by3 rinses in SDW for 1 min each. The surface sterilized roots were cutinto pieces (about 1-2 cm long) using sterile scissors and dropped intotest tube containing NDSM medium (Eckford et al., 2002). After 2-4 daysincubation at room temperature the tubes were observed and obviouspellicles drawn off and purified by subculture on R2A agar (Difco).

The roots from one portion were combined in a plastic bag and crushedwithin the bag with 10 mls of water added to suspend the root material.Pieces of crushed root were retrieved and either placed on PDA plates,or embedded in molten PDA at 45° C. Ten-fold serial dilutions of thesuspension were prepared in SDW and used to prepare spread plates on R2Aagar (Difco). R2A and PDA plates were incubated at 25° C. and examinedunder a dissecting microscope after 24-72 hours incubation. Colonieswere assessed for abundance, grouped according to morphology andrepresentative isolates were picked and streaked for purity onto freshR2A or PDA plates. Standard methods were used to identify isolates tospecies level by DNA extraction, PCR amplification and sequencing of 16SrDNA (bacteria) or ITSS region (fungi).

Microbial Evaluation

Two rounds of microbial evaluation were performed on isolates selectedon the basis of abundance, diversity, and species characteristics. Inthe first evaluation round, 80 treatments were tested comprising 68individual isolates and 12 consortia.

Selected bacterial and fungal isolates were cultured on R2A and PDAplates respectively and suspensions prepared in SDW for inoculation ofseeds as generally described in example 4.

The suspensions were diluted to 1×10⁷ (bacteria) and 1×10³ (fungi) perml for use as individual treatments. Consortia were prepared using equalvolumes of each individual microbial suspension. Basil seeds were soakedfor one hour in microbial suspensions then planted into 28 ml tubescontaining commercial potting mix (described in example 4) that had beenmoistened with 6 ml of tap water. Two ml of microbial suspension waspipetted over the top of each seed. Thirty replicates were prepared foreach treatment and 45 replicates were prepared for the no-microbecontrol.

Thirteen DAS 15 plants from each treatment and 22 no-microbe controlswere selected for harvest and WSC determination. Sample preparation wasperformed as described previously with the exception that after beadbeating, 0.8 ml of SDW was added to each tube and a second round of beadbeating was performed. A 0.5 mL sample of the resulting mixed suspensionwas then transferred to a 96-well microtube dilution block and stored at−20° C. Blocks were thawed and assayed for carbohydrate as previouslydescribed.

A total of 36 microbial treatments yielded median carbohydrateconcentrations greater than microbe-free controls. This data was used togenerate a refined set of 44 treatments comprising 34 individualisolates and 10 consortia for a second round of microbial evaluation.Treatments were selected on the basis of results for increased WSC andincluded individual isolates that performed well in consortia, as wellas new consortia prepared from highly ranked microbes.

Microbial treatments were prepared and the basil seed was soaked andplanted as described above with the exception that the number oftreatment replicates was increased to 45 and no-microbe controlsincreased to 90.

All plants were harvested 14 days after sowing and processed for WSCanalysis as described above, with the exception that blocks were frozenovernight after the first 30 minute heating step. Samples were thenthawed and processed as previously described. A dilution series of asingle basil sample was loaded onto all blocks to serve as an internalcontrol and enable normalisation of between-block variation.

A total of 20 microbial treatments yielded median WSC concentrationsgreater than microbe-free controls with 11 treatments yielding greaterthan 5% increases over the control (IOC; Table 3).

The treatment yielding the highest median carbohydrate concentration wasa new microbial consortium of the three top-ranking individual isolatesfrom the first round of microbial evaluation.

TABLE 3 Microbial treatments yielding carbohydrate concentrationsgreater than microbe-free controls in round 2 microbial evaluation.Treatment BDNZ# ID % IOC 60706, 60784, Sphingomonas mali, Flavobacterium14.0 61090 micromati, Penicillium sp. 60695, 60696, Sphingobiumchlorophenolicum, Massilia 12.4 60697, 60698, niastensis, Flavobacteriumlimicola, 60699, 60700 Rhizobium alamii, Sphingopyxis sp., Pelomonasaquatica 60587 Azospirillum lipoferum 11.4 60732, 60739, Mesorhizobiumamorphae, Asticcacaulis 10.6 60740, 60744, taihuensis, Ralstoniasolanacearum, 61082 Microbacterium foliorum, Trichoderma 60805Burkholderia megapolitana 10.2 60732 Mesorhizobium amorphae 7.9 61043Umbelopsis sp 7.5 60734 Aquabacterium fontiphilum 7.2 60797Rhodanobacter terrae 7.1 60706 Sphingomonas mali 7.1 60578, 60580,Sphingobium xenophagum, Pseudomonas 5.0 60696, 60697, moraviensis,Massilia niastensis, 61043 Flavobacterium limicola, Umbelopsis sp.No-microbe control 0.0 ID-putative identification based on closest matchin NCBI and/or RDPII databases

These results provide evidence that the method for directed selection ofmicrobes, also referred to herein as accelerated microbial selection,described by the present disclosure is capable of producing a set ofmicrobes that improve the production of water soluble carbohydrate inbasil.

Furthermore, as indicated by Table 3, the methods are able to identifymicrobial consortia that significantly improve the concentration ofwater soluble carbohydrate in basil.

Example 6: Identification of Endophytic Microbes that Improve the Growthof Maize (Zea mays)

Endophytic microbes are closely associated with or contained withinplant tissues, therefore may be less exposed to competition andstressors than microbes associated with the plant rhizosphere. It wouldbe desirable to create a group of endophytic microbes that are capableof promoting maize growth by means such as increasing plant biomass orgrain yield. In this example an endophytic microbe is defined as onethat is still viable after surface sterilisation of maize plant tissueswith 6.6% Dettol® (active ingredient: chloroxylenol 4.8%) for 1 minute.

Seventy-three soil samples from the North Island of New Zealand wereused as the source of microbial diversity. Soil samples (treatments)were mixed with sterile sand:vermiculite (1:1 or 1:2) as required toincrease drainage and volume. The resulting mixtures were placed in 28ml tubes and planted with 15 replicates of maize (Pioneer Zea mayshybrid seeds 37Y12) in each treatment. Seedlings were watered with amisting hose until germinated, then showered to saturation three timesweekly with additional watering as required. For remaining standardgrowing conditions see Table 1.

Three plants from each treatment were selected at 60 days after sowing(DAS). The stems of the maize plants were cut 5 cm above the soil anddiscarded. The roots and attached stems were shaken free of soil, washedto remove soil fragments and drained before the roots and stems werecombined in plastic bags. This material was then crushed within the bagwith 10 ml of water added to suspend the root material. The liquidportion of the resulting suspension was used as the microbial inoculumfor a non-selective enrichment round. The purpose of this extra roundwas to increase the abundance of microbes growing within maize tissues.Surface-sterilised maize (37Y12) seeds were soaked for one hour in 1 mlof the root suspension for each sample. Soaked seeds were then plantedinto 28 ml tubes (15 reps for each treatment) containing sterile sandand vermiculite 1:2 moistened with 6 ml Phostrogen® soluble plant food(diluted 1/450 v/v in sterile distilled water). The remaining rootsuspension was made up to a final volume of 40 ml using steriledistilled water (SDW) and 2 ml was pipetted over the planted seeds.

Round 1 Selection

Sixty days after sowing (DAS) the five largest plants in each treatmentwere selected and processed to provide the microbial inoculum for thefirst round of selection. The foliage of each of the selected plants wascut 5 cm above substrate level and discarded. The remaining basal stemand roots were washed thoroughly in tap water to remove any adherentsoil and then combined within treatments in plastic bags before beingsurface sterilised with 6.6% Dettol® for 1 minute to select forendophytic microbes. Roots were then rinsed 3 times in SDW for 1, 5 then10 minutes with agitation. Rinsed roots were crushed within the plasticbags as described above, and suspended in a final volume of 20 ml SDW.The resulting suspension was used to inoculate 15 surface-sterilisedmaize seeds (Pioneer Zea mays P9400) by soaking them for one hour in 10ml of the inoculum before they were planted into sterilesand:vermiculite 1:3 moistened with sterile synthetic fertiliser(Fahraeus, 1957). The remaining suspension was made up to a final volumeof 40 ml for each treatment and 2 ml was pipetted over the top of eachplanted seed. Thirty replicate tubes of microbe-free control seeds weresoaked in SDW and pipetted with 2 mls of water per tube in a duplicateprocess free of microbial inoculum. After planting the seeds werecovered with fresh dry substrate. Pots were watered with SDW for thefirst week after planting to maintain sterile conditions, then with tapwater three times weekly.

Round 2 Selection

Plants were harvested at 26 DAS. Foliage was cut and weighed asdescribed above. The remaining basal stem and roots of each plant wererinsed clean, blotted dry with fresh paper towels then weighed andbagged individually. The inoculum for the second round of selection wasprepared from the 20 treatments yielding the greatest mean biomass andthe five largest individual plants from all treatments. The roots andbasal stems of the 10 largest plants from each selected treatment werepooled, surface sterilised and crushed as described above. The fivelargest individual plants were processed individually as above. Thirtyreplicates (Pioneer P9400 seeds) were planted for each of the 25treatments in sterile sand:vermiculite 1:3 moistened with sterilesynthetic fertiliser.

Round 3 Selection

Plants were harvested at 26 DAS and processed as described previously.The six largest plants from the 7 treatments yielding the greatest meanbiomass were selected to create the inoculum for the third round ofselection. Plants were grown for 28 days, harvested and assessed asdescribed for previous rounds. The roots and basal stems of the threelargest plants from the top three treatments were pooled, and the twolargest plants in the experiment were selected individually to provideinoculum for microbial isolation.

Microbial Isolation

Microbial isolations were performed on root suspensions used toinoculate the R3 selection and on the suspensions prepared from the R3plants selected above. Bacterial and fungal isolations were performed asgenerally described above using R2A, PDA and NDSM media. A selectiveisolation step for actinomycetes was performed in which ethanol wasadded to the root suspension at a final concentration of 25%, incubatedat RT for 30 min then plated on R2A. Plates were examined after 1-7 daysincubation at 25° C. Colonies were assessed for abundance, groupedaccording to morphology and representative isolates were picked andsubcultured on to R2A. Standard methods were used to identify isolatesto species level by DNA extraction, PCR amplification and sequencing of16S rRNA gene (bacteria) or ITSS regions (fungi).

Microbial Evaluation Rounds

Two rounds of microbial evaluation were performed. In the firstevaluation round 79 strains were selected based on abundance, diversityand species characteristics. Bacterial isolates were prepared and usedto inoculate surface-sterilised seeds as described in example 4, withthe exception that maize seeds (P9400) were used. Fungal strains wereplated on PDA, incubated at 25° C. for 7 days then scraped off plateswith 5-10 ml SDW and sieved through a tea strainer to remove clumps ofmycelia and pieces of attached agar. The number of spores/hyphae wasdetermined using a Neubauer improved haemocytometer and compoundmicroscope and a dilution series of 5×10², 1×10³ and 2×10³ was prepared.Each dilution was then pipetted over 10 planted seeds thereby totalling30 seeds per replicate each at 3 dose levels. For both fungi andbacteria, surface-sterilised maize seeds (Pioneer P9400) were planted in28 ml tubes containing sterile potting mix (40% peat, 30% composted pinebark, 30% fine pumice, adjusted to pH 6.1 with lime) moistened withFahraeus solution (Fahraeus, 1957) before being covered with fresh drysubstrate. All plants were subsequently watered with tap water 3 timesweekly.

Plants were harvested 24 DAS and both foliage and roots were weighed.Microbial isolates yielding an average increase in foliar and/or rootweight over microbe-free controls were selected for a second round ofevaluation. The chosen strains were processed and planted as describedabove, with the exception that seeds were soaked and inoculated withfungal strains at a concentration of 1×10³ rather than three dilutionsand 15 replicates were planted for all strains. Foliage and roots wereharvested and weighed at 20 DAS. The results are shown in Table 4. Fourof the isolates resulted in significantly higher biomass than themicrobe-free controls.

TABLE 4 Endophytic microbes producing increased maize biomass % IOC %IOC % IOC BDNZ# Count FW RW BM ID 57119 12 13.1 9.9 11.8 Herbaspirillumfrisingense 57583 14 14.3 5.1 10.6 Acinetobacter sp. 57122 15 9.6 12.210.6 Xanthomonas translucens 57115 14 14.4 3.9 10.2 Pseudomonasmarginalis 57535 12 10.7 8.9 10.0 Herbiconiux ginsengi 57148 12 10.0 9.89.9 Burkholderia cepacia 57531 14 6.4 14.7 9.7 Microbacterium oxydans57150 14 11.8 6.1 9.5 Pseudomonas moraviensis 57597 15 9.5 8.6 9.1Azotobacter chroococcum 57155 14 7.8 10.6 8.9 Pseudomonasfrederiksbergensis 57154 15 6.6 8.4 7.3 Sphingomonas rosa 57602 14 6.28.6 7.2 Rhizobium endophyticum 57619 15 8.5 4.8 7.0 Bacillus thioparans57127 15 4.0 11.0 6.8 Terriglobus roseus 57612 15 8.2 3.9 6.5Novosphingobium rosa 58016 14 4.7 8.6 6.2 Azospirillum lipoferum 5756512 5.6 5.1 5.4 Streptomyces thermocarboxydus 57613 15 7.3 1.6 5.0Herbaspirillum frisingense Italics indicate a significant differencefrom microbe-free control(Fisher’s LSD); % IOC, percentage increase overcontrols Putative ID based on closest match in RDPII database to partial16S rRNA sequence

These results provide evidence that the method for directed selection ofmicrobes, also referred to herein as accelerated microbial selection,described by the present disclosure is capable of producing a set ofendophytic microbes that improve the growth of maize.

Furthermore, the microbes provided in Table 4 can be utilized toidentify microbial consortia that are capable of improving the growth ofmaize.

The disclosure has been described herein, with reference to certainembodiments, in order to enable the reader to practice the disclosurewithout undue experimentation. However, a person having ordinary skillin the art will readily recognise that many of the components andparameters may be varied or modified to a certain extent or substitutedfor known equivalents without departing from the scope of thedisclosure. It should be appreciated that such modifications andequivalents are herein incorporated as if individually set forth. Inaddition, titles, headings, or the like are provided to enhance thereader's comprehension of this document, and should not be read aslimiting the scope of the present disclosure.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

Throughout this specification and any claims which follow, unless thecontext requires otherwise, the words “comprise”, “comprising” and thelike, are to be construed in an inclusive sense as opposed to anexclusive sense, that is to say, in the sense of “including, but notlimited to”.

BIBLIOGRAPHY

-   Pikovskaya R I (1948). Mobilization of phosphorus in soil connection    with the vital activity of some microbial species. Microbiologia    17:362-370, incorporated by reference herein.-   Miche, L and Balandreau, J (2001). Effects of rice seed surface    sterilisation with hypochlorite on inoculated Burkholderia    vietamiensis. Appl. Environ. Microbiol. 67(7): p3046-3052,    incorporated by reference herein.-   Fahraeus, G. (1957). J. Gen Microbiol. 16: 374-381, incorporated by    reference herein.-   Ruth Eckford, R., Cook, F. D., Saul, D., Aislabie J., and J.    Foght (2002) Free-living Heterotrophic Bacteria Isolated from    Fuel-Contaminated Antarctic Soils. Appl. Environ. Microbiol    68(10):5181. Yemm and Willis (Biochem. J. 1954, 57: 508-514),    incorporated by reference herein.-   Colby, R. S., “Calculating Synergistic and Antagonistic Responses of    Herbicide Combinations, 1967 Weeds, vol. 15, pp. 20-22, incorporated    by reference herein.-   PCT/NZ2012/000041, filed Mar. 16, 2012, (WO2012/125050A1),    incorporated by reference herein.

1.-84. (canceled)
 85. A method for producing a microbial compositionthat promotes the development of a beneficial phenotypic plant trait,comprising: (a) growing a plurality of plants in a growth medium, in thepresence of a first microbial community; (b) selecting at least oneplant from said plurality of plants, following step (a), based upon abeneficial phenotypic trait exhibited by the plant relative to otherplants of said plurality; (c) acquiring a second microbial communityfrom the at least one plant selected in step (b); (d) repeating steps(a) to (c) in an iterative manner, wherein the second microbialcommunity acquired in step (c) is used as an inoculum in step (a) of anysuccessive repeat; (e) acquiring a microbial community that isassociated with promoting the development of a beneficial phenotypicplant trait and placing said microbial community in a medium; (f)separating the microbial community from the medium after a period oftime to provide one or more microbial composition substantially free ofmicroorganisms; and (g) applying the one or more composition of (f) to aplant or plant part, wherein the plant or plant part exhibits abeneficial phenotypic trait, as compared to a plant or plant part nothaving said microbial composition applied.
 88. The method according toclaim 85, wherein the microbial composition is formulated as a liquid.89. The method according to claim 85, wherein the microbial compositionis formulated as a solid.
 90. The method according to claim 85, whereinthe microbial composition is formulated as a seed coating.
 91. Themethod according to claim 85, wherein the microbial composition isformulated for application onto a growing plant.
 92. The methodaccording to claim 85, wherein the microbial composition is formulatedfor application on a fruit of the plant.
 93. The method according toclaim 85, wherein the microbial product comprises an agriculturallyacceptable auxiliary compound.
 94. The method according to claim 85,wherein the plurality of plants are members a fruit crop.
 95. The methodaccording to claim 85, wherein the plurality of plants are members of agenus selected from the group consisting of: Zea, Sorghum, Panicum,Oryza, Triticum, Hordeum, Secale, Triticale, or Avena.
 96. The methodaccording to claim 85, wherein the plurality of plants are legumes. 97.The method according to claim 85, wherein the plurality of plants arecotton plants.
 98. The method according to claim 85, wherein theplurality of plants are soybean plants.
 99. The method according toclaim 85, wherein the plurality of plants are Zea mays plants.
 100. Themethod according to claim 85, wherein the second microbial communityacquired in step (c) is from a root, stem, and/or foliar tissue from theat least one plant.
 101. The method according to claim 85, wherein thesecond microbial community acquired in step (c) is not acquired fromsoil surrounding the at least one plant.
 102. The method according toclaim 85, wherein the second microbial community acquired in step (c) isnot acquired from a root nodule from the at least one plant.
 103. Themethod according to claim 85, wherein the second microbial communityacquired in step (c) is acquired from above-ground tissue from the atleast one plant.
 104. The method according to claim 85, wherein thesecond microbial community acquired in step (c) is acquired frombelow-ground tissue from the at least one plant.
 105. The methodaccording to claim 85, wherein the second microbial community acquiredin step (c) is fractionated or normalised to adjust the ratio ofmicrobial organisms present therein.
 106. The method according to claim85, wherein the at least one plant selected in step (b) exhibits asuperior beneficial phenotypic trait, relative to other plants of saidplurality, selected from the group consisting of: growth rate, height,weight, production of metabolite, production of protein, production ofcarbohydrate, production of oil, leaf size, stem size, general health,survivability, speed of seed germination, quantity of biomass produced,root growth, leaf growth, shoot growth, grain yield, fibre yield, oilyield, seed yield, stress response, and/or combinations thereof. 107.The method according to claim 85, wherein said first microbial communityin step a) is obtained from a pre-existing microbial collection,database, library, or depository.